Fermented beverages and methods of production thereof

ABSTRACT

Provided herein are fermented beverages comprising symbiotic microbial communities, and methods of production thereof.

RELATED APPLICATION

This application is a continuation of U.S. non-provisional applicationSer. No. 17/515,358, filed on Oct. 29, 2021, which claims the benefitunder 35 U.S.C. 119(e) of U.S. provisional application No. 63/108,189,filed Oct. 30, 2020, both of which are incorporated by reference hereinin their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Award Number1940409 from the National Science Foundation. The government has certainrights in the invention.

BACKGROUND OF INVENTION

Kombucha is a fermented beverage produced by fermenting sweetened teausing a symbiotic culture of bacteria and yeast, referred to as a SCOBY.The composition of bacterial and yeast strains in a SCOBY used toproduce fermented products varies and is typically serially passagedbetween fermentation batches. Such passaging leads to potentialvariation between batches and the resulting fermented beverage.Consumption of kombucha and the availability of commercial kombucha andother fermented products has recently increased and is thought to beassociated with a range of beneficial health effects, such as aiding indigestion, metabolism, immunity, liver function, through deliveringliving microbial cultures.

SUMMARY OF INVENTION

Aspects of the present disclosure provide fermented beverages comprisinga symbiotic microbial community comprising at least one bacterial strainhaving a 16S rDNA sequence comprising at least 95% sequence identity toa nucleic acid sequence provided by SEQ ID NOs: 1-3, and at least onebacterial strain having a16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs:4-7, and at least one additional microbial strain, a sugar content thatis less than 20 grams per liter (g/L), and an ethanol level that is lessthan 0.5% alcohol by volume (abv, v/v).

In some embodiments, the symbiotic microbial community comprises atleast two, at least three, or at least four additional microbialstrains. In some embodiments, at least one of the additional microbialstrains is a bacterial strain. In some embodiments, each of theadditional microbial strains is a bacterial strain. In some embodiments,the additional bacterial strain belongs to the genus Lactobacillus,Gluconobacter, Leuconostoc, Acetobacter, Hafnia/Obesumbacterium,Lactococcus, Pediococcus, or Bacillus. In some embodiments, theadditional bacterial strain belongs to the species Acetobacterpasteurianus, A. ghanesis, A. orientalis, A. tropicalis, Gluconobacteroxydans, G. roseus, G. japonicus, G. frateurii, Leuconostocmesenteroides, Lactobacillus senmaizukei, L. brevis, L. parakefiri, L.hilgardii, L. diolivorans, L. rapi, L. kisonesis, L. buchneri, L.fuchuensis, L. plantarum, L. paraplantarum, L. fabifermentans, L.pentosus, L. graminis, L. composti, Bacillus zanthoxyli, B. qingshengii,B. aryabhattai, B. flexus, B. megaterium, Hafnia alvei, Obesumbacteriumproteus, Lactococcus taiwanensis, L. lactis, Lactobacillus casei, L.paracasei, Pediococcus claussenii, P. stilesii, P. pentosaceus, P.acidilactici, Gluconacetobacter liquefaciens, Lactobacillus cerevisiae,L. kefiri, L. sunkii, L. otakiensis, L. parabuchneri, Leuconostoclactis, L. palmae, L. holzapfelii, L. citreum, Lactobacillus nagelii, L.satsumensis, Acetobacter papayae, A. suratthaniensis, A. peroxydans,Gluconacetobacter takamatsuzukensis, or G. asukensis. In someembodiments, the additional bacterial strain has a 16S rDNA sequencecomprising at least 95% sequence identity to a nucleic acid sequenceprovided by SEQ ID NOs: 8-19.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 2,4, 5, 6, 7, and 20-24. In some embodiments, the symbiotic microbialcommunity consists of bacterial strains having a 16S rDNA sequencecomprising at least 95% sequence identity to a nucleic acid sequenceprovided by SEQ ID NOs: 2, 5, 6, and 7.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 2,4, and 5. In some embodiments, the symbiotic microbial communityconsists of bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 2, 4, and 5.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 21,20, 22, and 24. In some embodiments, the symbiotic microbial communitycomprises bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 21, 23, and 20.

In some embodiments, the symbiotic microbial community consists ofbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 21,20, 22, and 24. In some embodiments, the symbiotic microbial communityconsists of bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 21, 23, and 20.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 2,4, 5, and 7. In some embodiments, the symbiotic microbial communityconsists of bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 2, 4, 5, and 7.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 21,23, 20, and 24. In some embodiments, the symbiotic microbial communityconsists of bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 21, 23, 20, and 24.

In some embodiments, the symbiotic microbial community does not comprisea yeast strain. In some embodiments, the symbiotic microbial communitycomprises at least 2×10⁷ colony forming units of each bacterial strainand each additional microbial strain per milliliter of the fermentedbeverage.

In some embodiments, the fermented beverage comprises a level of aceticacid less than 1 gram per liter (g/L). In some embodiments, thefermented beverage comprises a level of organic acid that is greaterthan 1 gram per liter (g/L), wherein the organic acid is not aceticacid. In some embodiments, the organic acid is lactic acid, gluconicacid, ketogluconic acid, or a combination thereof.

In some embodiments, the fermented beverage is shelf stable for at least2 weeks at a temperature of about 20° C. In some embodiments, thefermented beverage is shelf stable for at least 1 week at a temperatureof about 40° C. In some embodiments, the pH of the fermented beverage isless than about 3.5.

In some embodiments, the fermented beverage is kombucha, seltzer, soda,gut shot, water kefir, jun, fruit juice, vegetable juice, ginger beer, aflavored water product, or a probiotic beverage. In some embodiments, atleast one of the microbial strains is derived from a fermented foodproduct. In some embodiments, each of the microbial strains is derivedfrom a fermented food product.

In some embodiments, the bacterial strains and the additional microbialstrains are live in the fermented beverage. In some embodiments, thefermented beverage further comprises one or more additional components.In some embodiments, the additional component is a vitamin, mineral, orflavoring additives. In some embodiments, the one or more additionalcomponent is selected from the group consisting of black tea, green tea,fruit juice, and vegetable juice.

Aspects of the present disclosure provide methods of producing afermented food beverage, comprising (i) providing a medium comprising afermentable sugar at an initial sugar level; (ii) adding a symbioticmicrobial community to the medium to produce a culture, wherein thesymbiotic microbial community comprises at least one bacterial strainhaving a 16S rDNA sequence comprising at least 95% sequence identity toa nucleic acid sequence provided by SEQ ID NOs: 1-3, and at least onebacterial strain having a16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs:4-7, and at least one additional microbial strain; and (iii) fermentingthe culture under conditions to produce a fermented beverage.

In some embodiments, the symbiotic microbial community comprises atleast two, at least three, or at least four additional microbialstrains. In some embodiments, at least one of the additional microbialstrains is a bacterial strain. In some embodiments, each of theadditional microbial strains is a bacterial strain. In some embodiments,the additional bacterial strain belongs to the genus Lactobacillus,Gluconobacter, Leuconostoc, Acetobacter, Hafnia/Obesumbacterium,Lactococcus, Pediococcus, or Bacillus. In some embodiments, theadditional bacterial strain belongs to the species Acetobacterpasteurianus, A. ghanesis, A. orientalis, A. tropicalis, Gluconobacteroxydans, G. roseus, G. japonicus, G. frateurii, Leuconostocmesenteroides, Lactobacillus senmaizukei, L. brevis, L. parakefiri, L.hilgardii, L. diolivorans, L. rapi, L. kisonesis, L. buchneri, L.fuchuensis, L. plantarum, L. paraplantarum, L. fabifermentans, L.pentosus, L. graminis, L. composti, Bacillus zanthoxyli, B. qingshengii,B. aryabhattai, B. flexus, B. megaterium, Hafnia alvei, Obesumbacteriumproteus, Lactococcus taiwanensis, L. lactis, Lactobacillus casei, L.paracasei, Pediococcus claussenii, P. stilesii, P. pentosaceus, P.acidilactici, Gluconacetobacter liquefaciens, L. cerevisiae, L. kefiri,L. sunkii, L. otakiensis, L. parabuchneri, Leuconostoc lactis, L.palmae, L. holzapfelii, L. citreum, Lactobacillus nagelii, L.satsumensis, Acetobacter papayae, A. suratthaniensis, A. peroxydans,Gluconacetobacter takamatsuzukensis, or G. asukensis. In someembodiments, the additional bacterial strain has a 16S rDNA sequencecomprising at least 95% sequence identity to a nucleic acid sequenceprovided by SEQ ID NOs: 8-19. In some embodiments, the symbioticmicrobial community comprises bacterial strains having a 16S rDNAsequence comprising at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 2, 4, 5, 6, 7, and 20-24.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 2,5, 6, and 7. In some embodiments, the symbiotic microbial communityconsists of bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 2, 5, 6, and 7.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 2,4, and 5. In some embodiments, the symbiotic microbial communityconsists of bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 2, 4, and 5.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 21,20, 22, and 24. In some embodiments, the symbiotic microbial communityconsists of bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 21, 20, 22, and 24.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 21,23, and 20. In some embodiments, the symbiotic microbial communityconsists of bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 21, 23, and 20.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 2,4, 5, and 7. In some embodiments, the symbiotic microbial communityconsists of bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 2, 4, 5, and 7.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs: 21,23, 20, and 24. In some embodiments, the symbiotic microbial communityconsists of bacterial strains having a 16S rDNA sequence comprising atleast 95% sequence identity to a nucleic acid sequence provided by SEQID NOs: 21, 23, 20, and 24.

In some embodiments, the symbiotic microbial community does not comprisea yeast strain. In some embodiments, the symbiotic microbial communitycomprises at least 2×10⁷ colony forming units of each bacterial strainand each additional microbial strain per milliliter of the fermentedbeverage.

In some embodiments, the fermented beverage comprises a level of aceticacid less than 1 gram per liter (g/L). In some embodiments, thefermented beverage comprises a level of organic acid that is greaterthan 1 gram per liter (g/L), wherein the organic acid is not aceticacid. In some embodiments, the organic acid is lactic acid, gluconicacid, ketogluconic acid, or a combination thereof.

In some embodiments, the fermented beverage is shelf stable for at least2 weeks at a temperature of about 20° C. In some embodiments, thefermented beverage is shelf stable for at least 1 week at a temperatureof about 40° C. In some embodiments, the pH of the fermented beverage isless than about 3.5.

In some embodiments, the fermented beverage is kombucha, seltzer, soda,gut shot, water kefir, jun, fruit juice, vegetable juice, ginger beer, aflavored water product, or a probiotic beverage.

In some embodiments, at least one of the microbial strains is derivedfrom a fermented food product. In some embodiments, each of themicrobial strains is derived from a fermented food product. In someembodiments, the bacterial strains and the additional microbial strainsare live in the fermented beverage.

In some embodiments, the fermented beverage further comprises one ormore additional components. In some embodiments, the additionalcomponent is a vitamin, mineral, or flavoring additives. In someembodiments, the one or more additional component is selected from thegroup consisting of black tea, green tea, fruit juice, and vegetablejuice. In some embodiments, the fermented beverage has a pH less thanabout 3.5.

In some embodiments, the fermenting is performed in a batch reactor. Insome embodiments, the fermenting is performed at about 18-37° C. In someembodiments, the fermenting is performed for at least 6 days at about20° C.

In some embodiments, the initial sugar level is 2.5-20 grams per liter(g/L) of a sugar source. In some embodiments, the initial sugar level isabout 10 grams per liter (g/L) of a sugar source. In some embodiments,the sugar source is a cane sugar, palm sugar, maple syrup, fruit juice,vegetable juice, brown sugar, molasses, agave nectar, honey, date syrup,date paste, date sugar, coconut sugar, or coconut water.

In some embodiments, the bacterial strains and the additional microbialstrain(s) replicate faster when in the symbiotic microbial communitycompared to when not in the symbiotic microbial community. In someembodiments, the bacterial strains and the additional microbialstrain(s) grow to a higher biomass when in the symbiotic microbialcommunity compared to when not in the symbiotic microbial community. Insome embodiments, the symbiotic microbial community comprises at least2×10⁵ colony forming units. In some embodiments, the biomass of thesymbiotic microbial community is stable over at least 60 days. In someembodiments, the symbiotic microbial community reduces or preventsgrowth of undesired microbial strains.

In some embodiments, the method further comprises carbonating thefermented beverage to produce a carbonated fermented beverage.

Aspects of the present disclosure provide fermented beverage obtained orobtainable by any of the methods described herein.

BRIEF DESCRIPTION OF DRAWINGS

It is to be understood that the Figures are not necessarily to scale,emphasis instead being placed upon generally illustrating the variousconcepts discussed herein.

FIG. 1A shows the pH of fermentation reactions performed using theindicated individual microbial strains or the microbial community(“Community”) at the indicated time points. FIG. 1B shows the pH offermentation reactions performed using the indicated individualmicrobial strains or the microbial community (“Community”) at day 3.Each plot shows mean and standard deviation of 3 biological replicates.Strain 5 corresponds to SEQ ID NO: 5; Strain 2 corresponds to SEQ ID NO:2; Strain 6 corresponds to SEQ ID NO: 6; and Strain 7 corresponds to SEQID NO: 7. Community corresponds to a microbial community containingStrains 5, 2, 6, and 7.

FIG. 2A shows the biomass (CFU/mL) of microbial strains in fermentationreactions performed using the indicated individual microbial strains orthe microbial community (“Community”) at the end of the fermentationprocess (day 0). FIG. 2B shows the biomass of microbial strains infermentation reactions performed using the indicated individualmicrobial strains or the microbial community (“Community”) 7 daysfollowing the end of fermentation (day 7). FIG. 2C shows the biomass ofmicrobial strains in fermentation reactions performed using themicrobial community (“Community”) as compared to individual Strain 7 at7 days following the end of fermentation (day 7). Each plot shows meanand standard deviation of 3 biological replicates. Strain 5 correspondsto SEQ ID NO: 5; Strain 2 corresponds to SEQ ID NO: 2; Strain 6corresponds to SEQ ID NO: 6; and Strain 7 corresponds to SEQ ID NO: 7.Community corresponds to a microbial community containing Strains 5, 2,6, and 7.

FIG. 3A shows the pH of fermentation reactions performed using themicrobial community and various medium formulations containing canesugar with ginger (“Ginger”) at the indicated initial sugar levels (5g/L, 10 gL, 15 g/L, and 20 g/L). FIG. 3B shows the pH of fermentationreactions performed using the microbial community and various mediumformulations containing apple fruit juice (“Apple”) at the indicatedinitial sugar levels (5 g/L, 10 gL, 15 g/L, and 20 g/L). Each plot showsmean and standard deviation of 3 biological replicates.

FIG. 4 shows the end biomass (CFU/mL) at the time of bottling ofmicrobial strains in fermentation reactions using the microbialcommunity and the medium formulations containing cane sugar with ginger(“Ginger”) or apple fruit juice (“Apple”) at the indicated initial sugarlevels (5 g/L, 10 gL, 15 g/L, and 20 g/L). The plot shows mean andstandard deviation of 3 biological replicates. The dotted linescorrespond to the equivalent to 1 billion CFUs per 8 oz serving or 10billion CFUs per 8 oz serving.

FIG. 5A shows terminal sugar levels of fermentation reactions using themicrobial community and medium formulations containing cane sugar withginger (“Ginger”) or apple fruit juice (“Apple”) at the indicatedinitial sugar levels (5 g/L, 10 g/L, 15 g/L, and 20 g/L) at the time ofbottling. FIG. 5B shows terminal sugar levels of fermentation reactionsusing the microbial community and medium formulations containing canesugar with ginger (“Ginger”) or apple fruit juice (“Apple”) at theindicated initial sugar levels 14 days after bottling. FIG. 5C shows thepercent of sugar removed from medium formulations containing cane sugarwith ginger (“Ginger”) or apple fruit juice (“Apple”) at the indicatedinitial sugar levels using the microbial communities at the time point14 days after bottling. FIGS. 5A and 5B show terminal sugar levels asthe total sugar per 8 oz serving. Each plot shows mean and standarddeviation of 3 biological replicates. The dotted line indicates 0.5 g,which corresponds to the threshold to be considered a “zero-sugar” foodor beverage product for the U.S. Food and Drug Administration.

FIG. 6 shows the pH stability of fermented products produced byfermentation reactions using the microbial community and mediumformulations containing cane sugar with ginger (“Ginger,” top panel) orapple fruit juice (“Apple,” bottom panel) indicated initial sugar levels(5 g/L, 10 g/L, 15 g/L, and 20 g/L). The fermented products were storedat 35° C. and assessed at the indicated time points. The plot shows meanand standard deviation of 3 biological replicates. The microbialcommunity contains Strains 5, 2, 6, and 7.

FIG. 7 shows the long term pH stability of fermented products producedby fermentation reactions using the microbial community and subsequentlystored at an elevated temperature (35° C.) or at room temperature (22°C.). The pH was assessed on each of the indicated days postfermentation. The plot shows mean and standard deviation of 3 biologicalreplicates. The microbial community contains Strains 2, 4, 5, and 7.

FIG. 8 shows the biomass (CFU/mL) at the time of bottling of microbialstrains in fermentation reactions using the microbial community and themedium formulations containing cane sugar with ginger (“Ginger”) at theindicated initial sugar levels (0.5%, 1%, 1.5%, and 2%) at the end offermentation (Day 0) or following storage at room temperature (22° C.)for 28 days (Day 28). The plot shows the mean and individual results of3 biological replicates. The microbial community contains Strains 2, 4,5, and 7.

FIG. 9 shows alcohol by volume (% ABV) of fermentation reactions usingthe microbial community and medium formulations containing cane sugarwith ginger (“Ginger”) or apple fruit juice (“Apple”) at the indicatedinitial sugar levels (5 g/L, 10 g/L, 15 g/L, and 20 g/L), as compared tothe negative control (assay blank). The % ABV was assessed at threeweeks after fermentation finished. The plot shows the mean andindividual results of 2 technical replicates. The microbial communitycontains Strains 2, 4, 5, and 7.

FIG. 10 shows the stability of the biomass (CFU/mL) in fermentationreactions performed using the microbial community (“Community”) ascompared to the individual strains at the indicated time pointspost-fermentation. Strain 5 corresponds to SEQ ID NO: 5; Strain 2corresponds to SEQ ID NO: 2; Strain 6 corresponds to SEQ ID NO: 6; andStrain 7 corresponds to SEQ ID NO: 7. Community corresponds to amicrobial community containing Strains 5, 2, 6, and 7. The plot showsthe mean and individual results of 3 biological replicates.

FIG. 11 shows the biomass (CFU/mL) of contaminant strain followinginoculation of Brettanomyces sp. into fermentation reactions performedusing the microbial community (“Community”) as compared to theindividual strains and a negative control (media alone). Strain 5corresponds to SEQ ID NO: 5; Strain 2 corresponds to SEQ ID NO: 2;Strain 6 corresponds to SEQ ID NO: 6; and Strain 7 corresponds to SEQ IDNO: 7. Community corresponds to a microbial community containing Strains5, 2, 6, and 7. The plot shows the mean and individual results of 4 or 5biological replicates.

DETAILED DESCRIPTION OF INVENTION

Kombucha and other fermented beverages, such as kefirs and ginger beers,that contain living cultures are often considered “health beverages” andalternatives to other non-alcoholic or low alcohol content beverages.However, these products, like many soda products, are frequently high insugar with upwards of 12-15 grams per serving. The fermentation processused for kombucha production is not highly scalable to large scalebioreactors and typically must be fermented in batch in smallreactors/containers. The microbial communities utilized to producekombucha are not defined and typically include hundreds of microbialstrains. These communities can vary batch to batch, resulting influctuations in taste, flavor and inability to scale production in arational and predictable manner. The organisms used to produce Kombuchaalso can result in final products with relatively high levels of alcoholthat may be higher than the U.S. federal limit for non-alcoholicproducts (i.e., 0.5% ABV).

Additionally, kombucha must be maintained at reduced (refrigerated)temperatures for storage prior to consumption. The need for cold-chaindistribution vastly increases the cost associated with commerciallyavailable kombucha products and limits the ability of manufacturers tosuccessfully compete in the soda market, which is estimated to be a muchlarger market by at least two orders of magnitude.

In addition, the fermentation process for producing kombucha involvesculturing a symbiotic culture of bacterial and yeast, referred to as aSCOBY, and passaging the SCOBY between serial fermentation batches. Afrequent product of the fermentation process using a SCOBY is aceticacid, which can impart an undesired flavor profile characterized as“vinegar-like.” This strong, sometimes off-putting, flavor may alsolimit consumer acceptability and correspondingly the size of the targetmarket.

The present disclosure is based, at least in part, on the development ofsymbiotic microbial communities and fermentation methods of using suchmicrobial communities to produce fermented products having desiredproperties. In contrast to typical microbial communities used in theproduction of fermented beverages such as kombucha as well as singlestrain fermentation approaches, the symbiotic microbial communitiesdescribed herein provide a number of advantages, including fermentingsugars more quickly; increased resistance to invasion of thefermentation culture by environmental pathogens/contaminants; impartingcomplex and appealing flavor profiles to the fermented beverage,including low to no acetic acid and high levels of lactic acid andgluconic acids; producing a fermented beverage with very low to noethanol in the finished product; and providing a diverse population ofliving microbial cultures in the fermented beverage for consumption. Thefermented products described herein may be characterized as having amore fruity and appealing taste profile as compared to vinegar-like andstrong flavor typically associated with kombucha products. Thesynergistic interactions between the microbial strains of the symbioticmicrobial communities described herein are particularly unexpected aseach of the microbial strains were obtained from and evolved indifferent naturally fermented food sources.

Provided herein are fermented beverages comprising a symbiotic microbialcommunity and having reduced sugar content and reduced levels ofalcohol. Also provided herein are methods of producing fermentedbeverages involving providing a medium comprising a fermentable sugarand adding any of the symbiotic microbial community described herein andfermenting the culture to produce a fermented beverage.

Symbiotic Microbial Communities

Aspects of the present disclosure provide symbiotic microbialcommunities, fermented beverages comprising such microbial communities,and methods of producing fermented beverages involving a fermentationprocess using the microbial communities. As used herein, the term“symbiotic microbial community” refers to a plurality of microbialstrains that when grown or cultured together have a symbioticrelationship, which leads to the production of a fermented producthaving desired properties. As used herein, the term “symbioticrelationship” refers to a relationship between two or more microbialstrains that have a positive interaction in which at least one of themicrobial strains benefits from the relationship. In some embodiments,the symbiotic relationship is a mutualistic relationship in which bothmicrobial strains (or in the context of more than two microbial strains,all microbial strains) benefit from the relationship.

In contrast to fermented beverages that are produced using naturallyoccurring microbial communities, such as a SCOBY, which are typicallyundefined communities and/or have varying microbial composition that maychange during passaging, the composition of the microbial communities ofthe present disclosure are defined and rationally designed to achievefermented beverages having desired properties (e.g., reduced sugarcontent, low alcohol content, complex flavor profiles, shelf stability).For example, the microbial species are obtained from distinct sourcesand relative amounts of each of the microbial strains are controlled andrelatively uniform between disparate fermentation batches.

In some embodiments, the symbiotic microbial communities describedherein comprise two or more microbial strains. In some embodiments, thecompositions described herein comprise at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or up to 20 totalmicrobial strains.

It will be appreciated that the terms “microbial strains,” “microbes,”“microbial cells” and “microorganisms” are used interchangeably herein.In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or more) of themicrobial strains of the symbiotic microbial community are bacterialstrains. In some embodiments, all of the microbial strains of thesymbiotic microbial community are bacterial strains. In someembodiments, one or more (e.g., 1, 2, 3, 4, 5, or more) of the microbialstrains of the symbiotic microbial community are yeast strains. In someembodiments, the symbiotic microbial community does not contain yeaststrains. In some embodiments, the symbiotic microbial community does notcontain a yeast strain of the Brettanomyces genus. In some embodiments,the symbiotic microbial community does not contain a yeast strain of theSaccharomyces genus. In some embodiments, the symbiotic microbialcommunity does not contain a yeast strain of the Starmerella genus.

The symbiotic microbial communities of the present disclosure compriseat least one microbial strain (e.g., 1, 2, 3, 4, 5, or more) having a16S rDNA sequence comprising at least 95% sequence identity to thenucleic acid sequence provided by SEQ ID NOs: 1-3 and at least onemicrobial strain (e.g., 1, 2, 3, 4, 5, or more) having a 16S rDNAsequence comprising at least 95% sequence identity to the nucleic acidsequence provided by SEQ ID NOs: 4-7. The symbiotic microbialcommunities of the present disclosure comprise at least one microbialstrain (e.g., 1, 2, 3, 4, 5, or more) having a 16S rDNA sequencecomprising at least 95% sequence identity to the nucleic acid sequenceprovided by SEQ ID NOs: 1-3 and one microbial strain having a 16S rDNAsequence comprising at least 95% sequence identity to the nucleic acidsequence provided by SEQ ID NOs: 4-7. The symbiotic microbialcommunities of the present disclosure comprise one microbial strainhaving a 16S rDNA sequence comprising at least 95% sequence identity tothe nucleic acid sequence provided by SEQ ID NOs: 1-3 and at least onemicrobial strain (e.g., 1, 2, 3, 4, 5, or more) having a 16S rDNAsequence comprising at least 95% sequence identity to the nucleic acidsequence provided by SEQ ID NOs: 4-7.

In some embodiments, the symbiotic microbial communities of the presentdisclosure consist of at least one microbial strain (e.g., 1, 2, 3, 4,5, or more) having a 16S rDNA sequence comprising at least 95% sequenceidentity to the nucleic acid sequence provided by SEQ ID NOs: 1-3 and atleast one microbial strain (e.g., 1, 2, 3, 4, 5, or more) having a 16SrDNA sequence comprising at least 95% sequence identity to the nucleicacid sequence provided by SEQ ID NOs: 4-7.

Microbial strains used to produce the fermented beverages in the methodsdescribed herein were classified into operational taxonomic units (OTUs)on the basis of sequence similarity. Representative sequences from eachOTU were compared with sequences in publicly available nucleic aciddatabases, such as Basic Local Alignment Search Tool (BLAST) todetermine closely related genera and species and were analyzed usingtaxonomic assignment tools, such as RDP Classifier, which assignbacterial taxonomy to representative sequences. Ribosomal 16S DNAsequences are provided below for representative microbial strains.

In some embodiments, the symbiotic microbial communities of the presentdisclosure comprise at least one microbial strain having a 16S rDNAsequence comprising at least 95% sequence identity to the nucleic acidsequence provided by SEQ ID NOs: 1-3 and at least one microbial strainhaving a 16S rDNA sequence comprising at least 95% sequence identity tothe nucleic acid sequence provided by SEQ ID NOs: 4-7 and at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, at least ten or more additionalmicrobial strains.

In some embodiments, the symbiotic microbial communities of the presentdisclosure consist of at least one microbial strain having a 16S rDNAsequence comprising at least 95% sequence identity to the nucleic acidsequence provided by SEQ ID NOs: 1-3 and at least one microbial strainhaving a 16S rDNA sequence comprising at least 95% sequence identity tothe nucleic acid sequence provided by SEQ ID NOs: 4-7 and at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, at least ten or more additionalmicrobial strains.

Any of the symbiotic microbial communities described herein may furthercomprise one or more additional microbial strain, such as a microbialstrain belonging to the genus Leuconostoc, Obseumbacterium, Lactococcus,Bacillus, Lactobacillus, Acetobacter, or Gluconobacter. In someembodiments, the symbiotic microbial communities of the presentdisclosure comprise at least one microbial strain having a 16S rDNAsequence comprising at least 95% sequence identity to the nucleic acidsequence provided by SEQ ID NOs: 1-3 and at least one microbial strainhaving a 16S rDNA sequence comprising at least 95% sequence identity tothe nucleic acid sequence provided by SEQ ID NOs: 4-7 and one or moreadditional microbial strain, such as a microbial strain belonging to thegenus Leuconostoc, Obseumbacterium, Lactococcus, Bacillus,Lactobacillus, Acetobacter, or Gluconobacter. In some embodiments, thesymbiotic microbial communities of the present disclosure comprise atleast one microbial strain having a 16S rDNA sequence comprising atleast 95% sequence identity to the nucleic acid sequence provided by SEQID NOs: 1-3 and 21 and at least one microbial strain having a 16S rDNAsequence comprising at least 95% sequence identity to the nucleic acidsequence provided by SEQ ID NOs: 4-7, 20, 22, 23, and 24 and additionalmicrobial strains belonging to the genus Leuconostoc, Obseumbacterium,Hafnia, Lactococcus, Bacillus, Lactobacillus, Acetobacter, Pediococcus,or Gluconobacter.

Any of the symbiotic microbial communities described herein may furthercomprise one or more additional microbial strain, such as a microbialstrain having a 16S rDNA sequence provided by any one of the nucleicacid sequences of SEQ ID NOs: 8-19. In some embodiments, the symbioticmicrobial communities of the present disclosure comprise at least onemicrobial strain having a 16S rDNA sequence comprising at least 95%sequence identity to the nucleic acid sequence provided by SEQ ID NOs:1-3 and 21 and at least one microbial strain having a 16S rDNA sequencecomprising at least 95% sequence identity to the nucleic acid sequenceprovided by SEQ ID NOs: 4-7, 20, 22, 23, and 24 and one or moreadditional microbial strain, such as a microbial strain having a 16SrRNA sequence provided by any one of the nucleic acid sequences of SEQID NOs: 8-19. In some embodiments, the symbiotic microbial communitiesof the present disclosure comprise at least one microbial strain havinga 16S rDNA sequence comprising at least 95% sequence identity to thenucleic acid sequence provided by SEQ ID NOs: 1-3 and 21 and at leastone microbial strain having a 16S rDNA sequence comprising at least 95%sequence identity to the nucleic acid sequence provided by SEQ ID NOs:4-7, 20, 22, 23, and 24 such as a microbial strain having a 16S rRNAsequence provided by any one of the nucleic acid sequences of SEQ IDNOs: 8-18.

Ribosomal 16S DNA sequences are provided below for representativemicrobial strains. The closest related identified bacterial species weredetermined based on whole genome sequence analysis and a combination ofanalysis of full length 16S rDNA sequences and the sequences of severalhousekeeping genes (e.g., dnaK, dnaJ, mutL) compared to publiclyavailable sequence databases. It should be appreciated that multiplebacterial strains disclosed herein may have the highest homology withthe same species. It should further be appreciated that the bacterialstrains disclosed herein that have a 16S rDNA sequence with a nucleicacid sequence selected from the group consisting of SEQ ID NOs:1-24, arealso homologous to other strains based on their whole genome sequence,or subset of their whole genome sequence.

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 1 (also referred to herein as “strain 1”) has the highest homologywith a bacterial strain of the species Acetobacter pasteurianus, A.ghanesis, A. orientalis, or A. tropicalis:

Strain 1 (SEQ ID NO: 1)TACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGTGTAGGCGGTTTGTACAGTCAGATGTGAAATCCCCGGGCTTAACCTGGGAGCTGCATTTGATACGTGCAGACTAGAGTGTGAGAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 2 (also referred to herein as “strain 2”) has the highest homologywith a bacterial strain of the species Gluconobacter oxydans or G.roseus.

Strain 2 (SEQ ID NO: 2)TACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGCGTAGGCGGTTGTTACAGTCACATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGATACGTGACGACTAGAGTTCGAGAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 3 (also referred to herein as “strain 3”) has the highest homologywith a bacterial strain of the species Gluconobacter japonicus, or G.frateurii.

Strain 3 (SEQ ID NO: 3)TACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGCGTAGGCGGTTGATGCAGTCAGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGAGACGCATTGACTAGAGTTCGAGAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 4 (also referred to herein as “strain 4”) has the highest homologywith a bacterial strain of the species Lactobacillus senmaizukei or L.brevis.

Strain 4 (SEQ ID NO: 4)TACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGGAGACTTGAGTGCAGAAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 5 (also referred to herein as “strain 5”) has the highest homologywith a bacterial strain of the species Leuconostoc mesenteroides.

Strain 5 (SEQ ID NO: 5)TACGTATGTCCCGAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTTATTAAGTCTGATGTGAAAGCCCGGAGCTCAACTCCGGAATGGCATTGGAAACTGGTTAACTTGAGTGCAGTAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 6 (also referred to herein as “strain 6”) has the highest homologywith a bacterial strain of the species Lactobacillus parakefiri, L.hilgardii, L. diolivorans, L. rapi, L. kisonesis, or L. buchneri.

Strain 6 (SEQ ID NO: 6) TACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACCGGGAGACTTGAGTGCAGAAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 7 (also referred to herein as “strain 7”) has the highest homologywith a bacterial strain of the species Lactobacillus fuchuensis, L.plantarum, L. paraplantarum, L. fabifermentans, L. pentosus, L.graminis, or L. composti.

Strain 7 (SEQ ID NO: 7) TACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 8 (also referred to herein as “strain 8”) has the highest homologywith a bacterial strain of the species Bacillus zanthoxyli, B.qingshengii, B. aryabhattai, B. flexus, or B. megaterium:

Strain 8 (SEQ ID NO: 8) TACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 9 (also referred to herein as “strain 9”) has the highest homologywith a bacterial strain of the species Hafnia alvei or Obesumbacteriumproteus.

Strain 9 (SEQ ID NO: 9) TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTGATTAAGTCAGATGTGAAATCCCCGAGCTTAACTTGGGAACTGCATTTGAAACTGGTCAGCTAGAGTCTTGTAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 10 (also referred to herein as “strain 10”) has the highest homologywith a bacterial strain of the species Lactococcus taiwanensis or L.lactis:

Strain 10 (SEQ ID NO: 10)TACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGTGGTTTATTAAGTCTGGTGTAAAAGGCAGTGGCTCAACCATTGTATGCATTGGAAACTGGTAGACTTGAGTGCAGGAGAG

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 11 (also referred to herein as “strain 11”) has the highest homologywith a bacterial strain of the species Lactobacillus casei, or L.paracasei.

Strain 11 (SEQ ID NO: 11)TACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 12 (also referred to herein as “strain 12”) has the highest homologywith a bacterial strain of the species Pediococcus claussenii, P.stilesii, P. pentosaceus, or P. acidilactici.

Strain 12 (SEQ ID NO: 12)TACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 13 (also referred to herein as “strain 13”) has the highest homologywith a bacterial strain of the species Gluconacetobacter liquefaciens.

Strain 13 (SEQ ID NO: 13)TACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGCGTAGGCGGTATGGACAGTCAGATGTGAAATTCCTGGGCTTAACCTGGGGGCTGCATTTGATACGTCCAAACTAGAGTGTGAGAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 14 (also referred to herein as “strain 14”) has the highest homologywith a bacterial strain of the species Lactobacillus cerevisiae.

Strain 14 (SEQ ID NO: 14)TACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGGTAACTTGAGTGCAGAAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 15 (also referred to herein as “strain 15”) has the highest homologywith a bacterial strain of the species Lactobacillus kefiri, L. sunkii,L. otakiensis, or L. parabuchneri.

Strain 15 (SEQ ID NO: 15)TACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACCAGGAGACTTGAGTGCAGAAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 16 (also referred to herein as “strain 16”) has the highest homologywith a bacterial strain of the species Leuconostoc lactis, L. palmae, L.holzapfelii, or L. citreum.

Strain 16 (SEQ ID NO: 16)TACGTATGTCCCGAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTGATTAAGTCTGATGTGAAAGCCCGGAGCTCAACTCCGGAATGGCATTGGAAACTGGTTAACTTGAGTGTTGTAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 17 (also referred to herein as “strain 17”) has the highest homologywith a bacterial strain of the species Lactobacillus nagelii or L.satsumensis.

Strain 17 (SEQ ID NO: 17)TACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAACGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGTCGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 18 (also referred to herein as “strain 18”) has the highest homologywith a bacterial strain of the species Acetobacter papayae, A.suratthaniensis, or A. peroxydans.

Strain 18 (SEQ ID NO: 18)TACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGTGTAGGCGGTTTTGACAGTCAGATGTGAAATCCCCGGGCTTAACCTGGGAGCTGCATTTGAGACGTTAAGACTAGAGTGTGAGAGA

The bacterial strain comprising a 16S rDNA sequence provided by SEQ IDNO: 19 (also referred to herein as “strain 19”) has the highest homologywith a bacterial strain of the species Gluconacetobactertakamatsuzukensis or G. asukensis:

Strain 19 (SEQ ID NO: 19)TACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGCGTAGGCGGTTTGGACAGTCAGATGTGAAATTCCTGGGCTTAACCTGGGGGCTGCATTTGATACGTCCAGACTAGAGTGTGAGAGA

It should further be appreciated that the bacterial species describedherein may be identified based on the nucleotide sequence of the fulllength 16S rDNA, as provided below. Alternatively or in addition, thebacterial species described herein may be identified based onidentification of 16S sequences through whole genome sequencing, and bycomparing the sequences with 16S databases, or comparing the wholegenome sequence, or a subset of their whole genome sequence to sequencedatabases.

Full length 16S rDNA sequences are provided below for representativespecies.

Strain 5 - 16S ribosomal DNA (SEQ ID NO: 20) - Leuconostoc mesenteroides.ATTGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCACAGCGAAAGGTGCTTGCACCTTTCAAGTGAGTGGCGAACGGGTGAGTAACACGTGGACAACCTGCCTCAAGGCTGGGGATAACATTTGGAAACAGATGCTAATACCGAATAAAACTTAGTGTCGCATGACACAAAGTTAAAAGGCGCTTCGGCGTCACCTAGAGATGGATCCGCGGTGCATTAGTTAGTTGGTGGGGTAAAGGCCTACCAAGACAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCTGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGTGTGATGAAGGCTTTCGGGTCGTAAAGCACTGTTGTATGGGAAGAACAGCTAGAATAGGAAATGATTTTAGTTTGACGGTACCATACCAGAAAGGGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCCCGAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTTATTAAGTCTGATGTGAAAGCCCGGAGCTCAACTCCGGAATGGCATTGGAAACTGGTTAACTTGAGTGCAGTAGAGGTAAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTTACTGGACTGCAACTGACGTTGAGGCTCGAAAGTGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAACACTAGGTGTTAGGAGGTTTCCGCCTCTTAGTGCCGAAGCTAACGCATTAAGTGTTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGAAGCTTTTAGAGATAGAAGTGTTCTCTTCGGAGACAAAGTGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCAGCATTCAGATGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGCGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGCGTATACAACGAGTTGCCAGCCCGCGAGGGTGAGCTAATCTCTTAAAGTACGTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAATGCCCAAAGCCGGTGGCCTAACCTTTTAGGAAGGAGCCGTCTAAGGCAGGACAGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTTStrain 2 - 16S ribosomal DNA (SEQ ID NO: 21) - Gluconobacter oxydansCTGAGAGTTTGATCCTGGCTCAGAGCGAACGCTGGCGGCATGCTTAACACATGCAAGTCGCACGAAGGTTTCGGCCTTAGTGGCGGACGGGTGAGTAACGCGTAGGGATCTATCCACGGGTGGGGGACAACTTCGGGAAACTGGAGCTAATACCGCATGATACCTGAGGGTCAAAGGCGCAAGTCGCCTGTGGAGGAACCTGCGTTCGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGATGATCGATAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTCGACGGGGACGATGATGACGGTACCCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGCGTAGGCGGTTGTTACAGTCAGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGATACGTGACGACTAGAGTTCGAGAGAGGGTTGTGGAATTCCCAGTGTAGAGGTGAAATTCGTAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCAACCTGGCTCGATACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTGTGCTGGATGTTGGGAAACTTAGTTTTTCAGTGTCGAAGCTAACGCGCTAAGCACACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGGGCTTGCATGGGGAGGACCGGTTCAGAGATGGACCTTTCTTCGGACCTCCCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCAGCACTTTCAGGTGGGCACTCTAGAGAGACTGCCGGTGACAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGTCCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGAAGCTACATGGTGACATGGTGCTGATCTCTAAAAGCCGTCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGGTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTCGACCTTAAGCCGGTGAGCGAACCGTAAGGACGCAGCCGACCACGGACGGGTCAGCGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTTTStrain 6 - 16S ribosomal DNA (SEQ ID NO: 22) - Lactobacillus parakefiriATGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGTCTTGGTCAATGATTTTAGGTGCTTGCACTTGACTGATTTGACATTGAGACGAGTGGCGAACTGGTGAGTAACACGTGGGTAACCTGCCCTTGAAGTAGAGGATAACACTTGGAAACAGGTGCTAATACTGCATAACAACGAAAACCGCCTGGTTTTCGTTTGAAAGATGGCTTCGGCTATCGCTTTAGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGATGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTGGAGAAGAACGGGTGTCAGAGTAACTGTTGACATCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACCGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAACCTAAGAGATTAGGCGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCAGCATTTAGTTGGGCACTCTAGCAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAAACCGCGAGGTCAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGGTAACCTTCGGGGGCCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTStrain 4 - 16S ribosomal DNA (SEQ ID NO: 23) - Lactobacillus brevisATGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCATGCCTAATACATGCAAGTCGAACGAGCTTCCGTTGAATGACGTGCTTGCACTGATTTCAACAATGAAGCGAGTGGCGAACTGGTGAGTAACACGTGGGGAATCTACCCAGAAGCAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAACAAAATCCGCATGGATTTTGTTTGAAAGGTGGCTTCGGCTATCACTTCTGGATGATCCCGCGGCGTATTAGTTAGTTGGTGAGGTAAAGGCCCACCAAGACGATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAATGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACACCTTTGAGAGTAACTGTTCAAGGGTTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTAGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAATCTTAGAGATAAGACGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTCAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAAGTCGTGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGATAACCTTCGGGAGTCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTStrain 7 - 16S ribosomal DNA (SEQ ID NO: 24) - Lactobacillus plantarumTTTGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCAGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAPAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT

It should be appreciated that the compositions may include multiplestrains of a particular species. For example, as shown in Table 2,several of the microbial communities contain more than one strainshaving the same or highly related 16S rDNA sequences. In someembodiments, the composition includes multiple strains of a particularspecies that are obtained from independent sources but the strains havethe same or highly related 16S rDNA sequences.

Aspects of the disclosure relate to microbial strains with 16S rDNAsequences that have sequence identity to a nucleic acid sequence of anyone of the sequences of the microbial strains or microbial speciesdescribed herein. The terms “identical,” or percent “identity,” in thecontext of two or more nucleic acids or amino acid sequences, refer totwo or more sequences or subsequences that are the same. Two sequencesare “substantially identical” if two sequences have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%,99.7%, 99.8% or 99.9% sequence identity) over a specified region of anucleic acid or amino acid sequence or over the entire sequence, whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Optionally, the identity exists over a region that is atleast about 50 nucleotides in length, or more preferably over a regionthat is 100 to 500 or 1000 or more nucleotides in length. In someembodiments, the recited degree of identity exists over the length ofthe 16S rRNA or 16S rDNA sequence.

In some embodiments, the microbial strain has at least 60%, at least70%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, atleast 99.9%, or up to 100% sequence identity relative to any of themicrobial strains or microbial species described herein over a specifiedregion (such as a region of the 16S rDNA sequences provided herein) orover the entire sequence (such as the entire 16S rDNA). It would beappreciated by one of skill in the art that the term “sequence identity”or “percent sequence identity,” in the context of two or more nucleicacid sequences or amino acid sequences, refers to a measure ofsimilarity between two or more sequences or portion(s) thereof. In someembodiments, the identity exists over the length of the 16S rRNA or 16SrDNA sequence.

Additionally, or alternatively, two or more sequences may be assessedfor the alignment between the sequences. An alignment of 100% or “totalalignment” referring to two or more nucleic acids or amino acidsequences, refers to two or more sequences or subsequences that are thesame. Two sequences are “substantially aligned” if two sequences haveless than 100% alignment, such as a specified percentage of amino acidresidues or nucleotides that are the same (e.g., at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical)over a specified region or over the entire sequence, when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.Optionally, the alignment exists over a region that is at least about 50nucleotides in length, or more preferably over a region that is 100 to500 or 1000 or more nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. Methods of alignment ofsequences for comparison, including measuring identity betweensequences, are well known in the art. See, e.g., by the local homologyalgorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.(1970) 48:443, by the search for similarity method of Pearson andLipman. Proc. Natl. Acad. Sci. USA (1988) 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group.Madison. Wis.), or by manual alignment and visual inspection (see. e.g.,Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons,Inc. (Ringbou ed., 2003)). Two examples of algorithms that are suitablefor determining percent sequence identity and sequence similarity arethe BLAST and BLAST 2.0 algorithms, which are described in Altschul etal., Nuc. Acids Res. (1977) 25:3389-3402; and Altschul et al., J. Mol.Biol. (1990) 215:403-410, respectively.

In some aspects, the present disclosure provides symbiotic microbialcommunities comprising at least one microbial strain belonging to thegenus Gluconobacter or Acetobacter and at least one microbial strainbelonging to the genus Lactobacillus. The combination of at least onemicrobial strain belonging to the genus Gluconobacter or Acetobacter andat least one microbial strain belonging to the genus Lactobacillus wereunexpectedly found to be symbiotic/synergistic when present together ina microbial community and also produce fermented products characterizedas particularly “fruity” and pleasing in sensory panels.

In some embodiments, the symbiotic microbial communities of the presentdisclosure comprise at least one microbial strain belonging to the genusGluconobacter or Acetobacter and at least one microbial strain belongingto the genus Lactobacillus, and at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least ten or more additional microbial strains.

In some embodiments, the symbiotic microbial communities of the presentdisclosure consist of at least one microbial strain belonging to thegenus Gluconobacter or Acetobacter and at least one microbial strainbelonging to the genus Lactobacillus and at least two, at least three,at least four, at least five, at least six, at least seven, at leasteight, at least nine, at least ten or more additional microbial strains.

Any of the symbiotic microbial communities described herein may furthercomprise one or more additional microbial strain, such as a microbialstrain belonging to the genus Leuconostoc, Obseumbacterium, Hafnia,Lactococcus, Bacillus, Lactobacillus, Acetobacter, Pediococcus, orGluconobacter. In some embodiments, the symbiotic microbial communitiesof the present disclosure comprise at least one microbial strainbelonging to the genus Gluconobacter or Acetobacter and at least onemicrobial strain belonging to the genus Lactobacillus and one or moreadditional microbial strain, such as a microbial strain belonging to thegenus Leuconostoc, Obseumbacterium, Hafnia, Lactococcus, Bacillus,Lactobacillus, Acetobacter, Pediococcus, or Gluconobacter. In someembodiments, the symbiotic microbial communities of the presentdisclosure comprise at least one microbial strain belonging to the genusGluconobacter or Acetobacter and at least one microbial strain belongingto the genus Lactobacillus and additional microbial strains belonging tothe genus Leuconostoc, Obseumbacterium, Hafnia, Lactococcus, Bacillus,Lactobacillus, Acetobacter, Pediococcus, or Gluconobacter.

Any of the symbiotic microbial communities described herein may furthercomprise one or more additional microbial strain, such as a microbialstrain belonging to the species Acetobacter pasteurianus, A. ghanesis,A. orientalis, A. tropicalis, Gluconobacter oxydans, G. roseus,Gluconobacter japonicus, G. frateurii, Lactobacillus senmaizukei, L.brevis, Leuconostoc mesenteroides, Lactobacillus parakefiri, L.hilgardii, L. diolivorans, L. rapi, L. kisonesis, L. buchneri, L.fuchuensis, L. plantarum, L. paraplantarum, L. fabifermentans, L.pentosus, L. graminis, L. composti, Bacillus zanthoxyli, B. qingshengii,B. aryabhattai, B. flexus, B. megaterium, Hafnia alvei, Obesumbacteriumproteus, Lactococcus taiwanensis, L. lactis, Lactobacillus casei, L.paracasei, Pediococcus claussenii, P. stilesii, P. pentosaceus, or P.acidilactici, Gluconacetobacter liquefaciens, Lactobacillus cerevisiae,L. kefiri, L. sunkii, L. otakiensis, L. parabuchneri, Leuconostoclactis, L. palmae, L. holzapfelii, or L. citreum, Lactobacillus nagelii,L. satsumensis, Acetobacter papayae, A. suratthaniensis, A. peroxydans,Gluconacetobacter takamatsuzukensis, or G. asukensis.

In some embodiments, the symbiotic microbial communities of the presentdisclosure comprise at least one microbial strain belonging to the genusGluconobacter or Acetobacter and at least one microbial strain belongingto the genus Lactobacillus and one or more additional microbial strain,such as a microbial strain belonging to the species Acetobacterpasteurianus, A. ghanesis, A. orientalis, A. tropicalis, Gluconobacteroxydans, G. roseus, Gluconobacter japonicus, G. frateurii, Lactobacillussenmaizukei, L. brevis, Leuconostoc mesenteroides, Lactobacillusparakefiri, L. hilgardii, L. diolivorans, L. rapi, L. kisonesis, L.buchneri, L. fuchuensis, L. plantarum, L. paraplantarum, L.fabifermentans, L. pentosus, L. graminis, L. composti, Bacilluszanthoxyli, B. qingshengii, B. aryabhattai, B. flexus, B. megaterium,Hafnia alvei, Obesumbacterium proteus, Lactococcus taiwanensis, L.lactis, Lactobacillus casei, L. paracasei, Pediococcus claussenii, P.stilesii, P. pentosaceus, or P. acidilactici, Gluconacetobacterliquefaciens, Lactobacillus cerevisiae, Lactobacillus kefiri, L. sunkii,L. otakiensis, L. parabuchneri, Leuconostoc lactis, L. palmae, L.holzapfelii, or L. citreum, Lactobacillus nagelii, L. satsumensis,Acetobacter papayae, A. suratthaniensis, A. peroxydans,Gluconacetobacter takamatsuzukensis, or G. asukensis.

In some embodiments, the symbiotic microbial communities of the presentdisclosure comprise at least one microbial strain belonging to the genusGluconobacter or Acetobacter and at least one microbial strain belongingto the genus Lactobacillus and additional microbial strains belonging tothe species Acetobacter pasteurianus, A. ghanesis, A. orientalis, A.tropicalis, Gluconobacter oxydans, G. roseus, Gluconobacter japonicus,G. frateurii, Lactobacillus senmaizukei, L. brevis, Leuconostocmesenteroides, Lactobacillus parakefiri, L. hilgardii, L. diolivorans,L. rapi, L. kisonesis, L. buchneri, Lactobacillus fuchuensis, L.plantarum, L. paraplantarum, L. fabifermentans, L. pentosus, L.graminis, L. composti, Bacillus zanthoxyli, B. qingshengii, B.aryabhattai, B. flexus, B. megaterium, Hafnia alvei, Obesumbacteriumproteus, Lactococcus taiwanensis, L. lactis, Lactobacillus casei, L.paracasei, Pediococcus claussenii, P. stilesii, P. pentosaceus, or P.acidilactici, Gluconacetobacter liquefaciens, Lactobacillus cerevisiae,Lactobacillus kefiri, L. sunkii, L. otakiensis, L. parabuchneri,Leuconostoc lactis, L. palmae, L. holzapfelii, or L. citreum,Lactobacillus nagelii, L. satsumensis, Acetobacter papayae, A.suratthaniensis, A. peroxydans, Gluconacetobacter takamatsuzukensis, orG. asukensis.

In some examples, the microbial community comprises Leuconostocmesenteroides subsp. suionicum, Gluconobacter oxydans, Lactobacillusparakefiri, and L. plantarum. In some examples, the microbial communityconsists of Leuconostoc mesenteroides subsp. suionicum, Gluconobacteroxydans, Lactobacillus parakefiri, and L. plantarum. A microbialcommunity containing Leuconostoc mesenteroides subsp. suionicum,Gluconobacter oxydans, Lactobacillus parakefiri, and L. plantarum isreferred to herein as “Community 1.” In some examples, the microbialcommunity comprises Leuconostoc mesenteroides subsp. suionicum,Gluconobacter oxydans, Lactobacillus parakefiri, and L. plantarum andone or more additional microbial strains. In some examples, themicrobial community comprises Leuconostoc mesenteroides subsp.suionicum, Gluconobacter oxydans, Lactobacillus parakefiri, and L.plantarum and does not contain a yeast strain.

As will be appreciated by one of ordinary skill in the art, nomenclatureregarding bacterial genus and species names may be reclassified toreflect phylogenetic relationships of microorganisms. See, e.g., Zhenget al. Inter. J. System. And Evol. Microbiol. (2020) 70(4).

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%(e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 5, 6, and 7. In someembodiments, the symbiotic microbial community consists of bacterialstrains having a 16S rDNA sequence comprising at least 95% (e.g., 95%,96%, 97%, 98%, 99% or higher) sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 2, 5, 6, and 7. In some embodiments,the symbiotic microbial community comprises bacterial strains having a16S rDNA sequence comprising a nucleic acid sequence provided by SEQ IDNOs: 2, 5, 6, and 7. In some embodiments, the symbiotic microbialcommunity consists of bacterial strains having a 16S rDNA sequencecomprising a nucleic acid sequence provided by SEQ ID NOs: 2, 5, 6, and7.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%(e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 20, 22, and 24. In someembodiments, the symbiotic microbial community consists of bacterialstrains having a 16S rDNA sequence comprising at least 95% (e.g., 95%,96%, 97%, 98%, 99% or higher) sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 21, 20, 22, and 24. In someembodiments, the symbiotic microbial community comprises bacterialstrains having a 16S rDNA sequence comprising a nucleic acid sequenceprovided by SEQ ID NOs: 21, 20, 22, and 24. In some embodiments, thesymbiotic microbial community consists of bacterial strains having a 16SrDNA sequence comprising a nucleic acid sequence provided by SEQ ID NOs:21, 20, 22, and 24.

In some examples, the microbial community comprises Leuconostocmesenteroides subsp. suionicum, Gluconobacter oxydans, and Lactobacillusbrevis. In some examples, the microbial community consists ofLeuconostoc mesenteroides subsp. suionicum, Gluconobacter oxydans, andLactobacillus brevis. A microbial community containing Leuconostocmesenteroides subsp. suionicum, Gluconobacter oxydans, and Lactobacillusbrevis is referred to herein as “Community 2.” In some examples, themicrobial community consists of Leuconostoc mesenteroides subsp.suionicum, Gluconobacter oxydans, and Lactobacillus brevis and one ormore additional microbial strains. In some examples, the microbialcommunity comprises Leuconostoc mesenteroides subsp. suionicum,Gluconobacter oxydans, and Lactobacillus brevis and does not contain ayeast strain.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%(e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 4, and 5. In some embodiments,the symbiotic microbial community consists of bacterial strains having a16S rDNA sequence comprising at least 95% (e.g., 95%, 96%, 97%, 98%, 99%or higher) sequence identity to a nucleic acid sequence provided by SEQID NOs: 2, 4, and 5. In some embodiments, the symbiotic microbialcommunity comprises bacterial strains having a 16S rDNA sequencecomprising a nucleic acid sequence provided by SEQ ID NOs: 2, 4, and 5.In some embodiments, the symbiotic microbial community consists ofbacterial strains having a 16S rDNA sequence comprising to a nucleicacid sequence provided by SEQ ID NOs: 2, 4, and 5.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%(e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 23, and 20. In someembodiments, the symbiotic microbial community consists of bacterialstrains having a 16S rDNA sequence comprising at least 95% (e.g., 95%,96%, 97%, 98%, 99% or higher) sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 21, 23, and 20. In some embodiments,the symbiotic microbial community comprises bacterial strains having a16S rDNA sequence comprising a nucleic acid sequence provided by SEQ IDNOs: 21, 23, and 20. In some embodiments, the symbiotic microbialcommunity consists of bacterial strains having a 16S rDNA sequencecomprising a nucleic acid sequence provided by SEQ ID NOs: 21, 23, and20.

In some examples, the microbial community comprises Leuconostocmesenteroides subsp. suionicum, Gluconobacter oxydans, Lactobacillusbrevis, and Lactobacillus plantarum. In some examples, the microbialcommunity consists of Leuconostoc mesenteroides subsp. suionicum,Gluconobacter oxydans, Lactobacillus brevis, and Lactobacillusplantarum. In some examples, the microbial community consists ofLeuconostoc mesenteroides subsp. suionicum, Gluconobacter oxydans,Lactobacillus brevis, and Lactobacillus plantarum and one or moreadditional microbial strains. In some examples, the microbial communitycomprises Leuconostoc mesenteroides subsp. suionicum, Gluconobacteroxydans, Lactobacillus brevis, and Lactobacillus plantarum, and does notcontain a yeast strain.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%(e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 4, 5, and 7. In someembodiments, the symbiotic microbial community consists of bacterialstrains having a 16S rDNA sequence comprising at least 95% (e.g., 95%,96%, 97%, 98%, 99% or higher) sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 2, 4, 5, and 7. In some embodiments,the symbiotic microbial community comprises bacterial strains having a16S rDNA sequence comprising a nucleic acid sequence provided by SEQ IDNOs: 2, 4, 5, and 7. In some embodiments, the symbiotic microbialcommunity consists of bacterial strains having a 16S rDNA sequencecomprising to a nucleic acid sequence provided by SEQ ID NOs: 2, 4, 5,and 7.

In some embodiments, the symbiotic microbial community comprisesbacterial strains having a 16S rDNA sequence comprising at least 95%(e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 23, 20, and 24. In someembodiments, the symbiotic microbial community consists of bacterialstrains having a 16S rDNA sequence comprising at least 95% (e.g., 95%,96%, 97%, 98%, 99% or higher) sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 21, 23, 20, and 24. In someembodiments, the symbiotic microbial community comprises bacterialstrains having a 16S rDNA sequence comprising a nucleic acid sequenceprovided by SEQ ID NOs: 21, 23, 20, and 24. In some embodiments, thesymbiotic microbial community consists of bacterial strains having a 16SrDNA sequence comprising a nucleic acid sequence provided by SEQ ID NOs:21, 23, 20, and 24.

The microbial strains of the symbiotic microbial communities may be in alive (e.g., viable) state or in an inactivated state (e.g., killed, notviable), or in a mixture of live and inactivated states. As will beappreciated by one of ordinary skill in the art, microbial cells may beconsidered live or living if the cells are metabolically active, e.g.,have a detectable level of metabolic activity. Being metabolicallyactive does not require proliferation or replication of the cells.Methods of evaluating whether a microbial strain is living and ismetabolically active are known in the art, for example, viabilityassays, detection of ATP measurements, membrane potential, respiratoryactivity, uptake of dyes. See, e.g., Emerson et al. Microbiome (2017)5:86. In some embodiments, one or more of the microbial strains of thesymbiotic microbial community are in a live state and one or more of themicrobial strains of the symbiotic microbial community are in aninactivated state (e.g., killed, not viable).

In some embodiments, the microbial strains of the symbiotic microbialcommunities provided herein are in vegetative form, meaning themicrobial cells are not actively growing and/or reproducing. In someembodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) of themicrobial strains of the symbiotic microbial community is in vegetativeform. In some embodiments, each of the microbial strains of thesymbiotic microbial community is in vegetative form. It will beappreciated that live microbial strains may be viable but may not beactively growing/replicating. For example, following utilization of thesugar source in a fermentation medium, microbial strains may slow orhalt active replication due to reduced levels of available nutrients.

In some embodiments, microbial strains of the symbiotic microbialcommunities provided herein are living and are alive in the fermentedproduct.

Viability can be determined by quantifying the colony forming units(CFU), for example by plating a sample of the microbial community, orfermented beverage, on a nutritive agar medium. The number of colonyforming units corresponds to the number of viable microbial cells in thesample tested such as the community or fermented beverage.

The microbial strains described herein may be obtained from or derivedfrom any source known in the art, such as from a food source or anenvironmental source. As used herein, the term “derived from” in thecontext of microbial strains derived from a particular source refers toobtaining a microbial strain from the source, which may involveisolating and/or propagating cells of a microbial strain. In someembodiments, the microbial strains are further manipulated, such aspurified and/or analyzed, prior to use in the fermented beverages andmethods described herein. As will be evident to one of ordinary skill inthe art, reference to a microbial strain or cells of a microbial strainthat is derived from a particular source encompasses progeny cellsthereof.

The microbial strains of the symbiotic microbial community may bederived from a fermented food or beverage, such as cultured milk andyogurt, natto, cheese, kombucha, wine, beer, cider, miso, kimchi,sauerkraut, fermented sausage, among others.

In some embodiments, at least one microbial strain of the symbioticmicrobial community may be purified. In some embodiments, at least onemicrobial strain of the symbiotic microbial community may be isolated.Any of the microbial strains described herein may be isolated and/orpurified, for example, from a source such as a food source (e.g., afermented food or beverage product) or an environmental source.

In some embodiments, the microbial community may be in a lyophilizedform. In some embodiments, at least one microbial strain of thesymbiotic microbial community is in a lyophilized form. In someembodiments, each of microbial strains of the symbiotic microbialcommunity is in a lyophilized form. In such embodiments, the microbialcommunity (or microbial strain thereof) may be rehydrated or suspendedand/or cultured prior to use in the methods described herein. In someembodiments, the microbial community (or microbial strain thereof) inlyophilized form is used directly, for example, without rehydrating orsuspension (e.g., directly added to a medium).

As used herein, the term “isolated” refers to a microbial cell ormicrobial strain that has been separated from one or more undesiredcomponent, such as other microbial cells or microbial strains, one ormore component of a growth medium, one or more component of a food orbeverage product, and/or one or more component of a sample, such as anenvironmental sample. In some embodiments, the microbial strains aresubstantially isolated from a source such that other components of thesource are not detected. In some embodiments, a microbial strain isisolated or purified from a sample and then cultured (grown, propagated)under the appropriate conditions for replication. The microbial strainthat is grown under appropriate conditions for replication cansubsequently be isolated/purified from the culture in which it is grown.

Also within the scope of the present disclosure are isolated microbialcommunities. In this context, the term “isolated” refers to a microbialcommunity that has been separated from one or more undesired component,such as other microbial cells, microbial strains, microbial communities,one or more component of a growth medium, one or more component of afood or beverage product, and/or one or more component of a sample, suchas an environmental sample. In some embodiments, the microbialcommunities are substantially isolated from a source such that othercomponents of the source are not detected. In some embodiments,microbial strains are individually cultured and then combined formingthe microbial community. In some embodiments, the microbial strains arecombined forming a microbial community, which is then cultured (grown,propagated) collectively, as a community under the appropriateconditions for replication. The microbial strain that is grown underappropriate conditions for replication can subsequently beisolated/purified from the culture in which it is grown.

The specific microbial strains selected and combined to form themicrobial communities described herein have been found to havebeneficial properties when present or used in combination, as comparedto microbial communities that contain different combinations ofmicrobial strains or to microbial strains alone (not in combination).

For example, the microbial communities described herein, when comparedto single strains or other microbial communities, may replicate morequickly and reach a higher biomass during fermentation. In someembodiments, the microbial communities described herein replicate at arate that is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, or up to 50-fold higher than a microbialcommunity containing a different combination of microbial strains or themicrobial strains when present or used alone under similar fermentationconditions. In some embodiments, the microbial communities describedherein replicate at a rate that is about 2-3-fold higher than amicrobial community containing a different combination of microbialstrains or the microbial strains when present or used alone undersimilar fermentation conditions.

In some embodiments, the microbial communities described herein reach abiomass that is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold,300-fold, 400-fold or up to 500-fold higher than the biomass that can bereached with a microbial community containing a different combination ofmicrobial strains or with a microbial strain alone under similarfermentation conditions. In some embodiments, the microbial communitiesdescribed herein reach a biomass that is about 5-10-fold higher than thebiomass that can be reached with a microbial community containing adifferent combination of microbial strains or with a microbial strainalone under similar fermentation conditions.

The microbial communities described herein are able to maintain arelatively stable biomass, for example in a fermented product, overtime. In some embodiments, the microbial communities described hereinare able to maintain a relatively stable biomass, for example in afermented product, over time as compared to the biomass of the microbialstrains when provided in the absence of the community (e.g.,individually). A microbial community is considered to be stable inmaintaining a biomass if there is less than 99% decrease (2-logs) in thebiomass over 60 days. In some embodiments, the microbial community has abiomass (CFU/mL) that decreases by less than 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%. 99%. 99.1%, 99.2%, 99.3%, 99.4%,99.5%, 99.6%, 99.7%, 99.8%, 99.8%, 99.9% or higher over a period of 60days, for example stored at room temperature. In some embodiments, themicrobial community has a biomass (CFU/mL) that decreases by less than10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%. 99%.99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.8%, 99.9% orhigher over a period of 60 days as compared to the biomass of thefermented beverage at a prior time point (e.g., at the end offermentation), another microbial community, or individual microbialstrains.

In some embodiments, the microbial community has a biomass (CFU/mL) thatdecreases by less than 1,000-fold (3 logs), 100-fold (2 logs), or lessthan 10-fold (1 log) over a period of 60 days, for example stored atroom temperature. In some embodiments, the microbial community has abiomass (CFU/mL) that decreases by less than 1,000-fold (3 logs),100-fold (2 logs), or less than 10-fold (1 log) over a period of 60days, for example as compared to the biomass of the fermented beverageat a prior time point (e.g., at the end of fermentation), anothermicrobial community, or individual microbial strains.

Methods of assessing the replication rate of a microbial strain orcommunity thereof, as well as methods of quantifying biomass, are knownin the art. See, e.g. Brown et al. Nature Biotechnology (2016) 34:1256-1263

As another example, the microbial communities described herein mayferment more quickly than other microbial communities or than singlemicrobial strains used alone. The rate of fermentation of a microbialcommunity can be assessed by any means known in the art, such as theutilization of a sugar source, level of a sugar in the fermentationmedium, change in pH of the medium, generation of a fermentation product(e.g., organic acids, CO₂). In some embodiments, the microbialcommunities described herein ferment at a rate that is at least1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, or up to 50-fold higher than a microbial communitycontaining a different combination of microbial strains or singlemicrobial strains when used alone under similar fermentation conditions.In some embodiments, the microbial communities described herein fermentat a rate that is about 2-3-fold higher than a microbial communitycontaining a different combination of microbial strains or singlemicrobial strains when used alone under similar fermentation conditions.

Contamination of fermentation reactions or products intended forconsumer consumption can result in spoilage of products, lost profit andrevenue for manufacturers, and represent a substantial health and safetyrisk to individuals who ingest contaminated products. The conditionsthat are critical for promoting fermentation, including availability ofsugar source and other nutrients and temperatures at which fermentationis typically performed are also conditions in which othermicroorganisms, including pathogenic microbial strains, survive andreplicate. Surprisingly, the microbial communities described herein havebeen found to be more “resilient” to invasion by environmentalcontaminants, including unwanted bacterial, yeast, and mold strains,such as Pseudomonas spp., Listeria monocytogenes, Salmonella spp.,Shigella flexneri, Escherichia coli, Vibrio cholerae, Bacillus spp.,Campylobacter jejuni, Candida spp., Cryptococcus spp., Debaryomycesspp., Hansenula spp., Pichia spp., Rhodotorula spp., Saccharomyces spp.,Torulopsis spp., Zygosaccharomyces spp., Dekkera spp., and Trichosporonspp.

The ability of a microbial community to resist, prevent, or minimizeinvasion by a contaminant, such as unwanted bacterial, yeast, and moldstrains, may result from active antimicrobial capacities (e.g.,bacteriocin production) of the individual microbial strains of thecommunity or the microbial community collectively. It should be notedthat production of inhibitory compounds, such as antimicrobialmolecules, by a microbial strain of the community may be stimulated byanother strain(s) of the community.

In some embodiments, the combination of microbial strains in thecommunity synergizes resulting in increased rate of fermentation and/ormicrobial replication, which may limit available nutrients forcontaminating strains, such as unwanted bacterial, yeast, and moldstrains. The capability of a microbial community (or individualmicrobial strains) to resist, prevent, or minimize invasion of afermented product or the fermentation process by a contaminant may beassessed by any means known in the art, such as by competition assays.

The microbial strains used in any of the symbiotic microbial communitiesdescribed herein are preferably “generally recognized as safe” (GRAS) orapproved as food additives according to the U.S. Food and DrugAdministration. See, e.g., Federal Food, Drug, and Cosmetic Act,sections 201(s) and 409.

Fermented Beverages

Aspects of the present disclosure relate to fermented beverages,including fermented beverages produced by any of the methods disclosedherein. The nature of the fermented beverage will depend on factors suchas the fermentable sugar provided for the fermentation process, thesugar source, as well as any additional components added prior to,during, or following fermentation. The fermented beverages describedherein are produced using symbiotic microbial communities that togetherlead to low sugar content, low alcohol content, and improved and desiredcharacteristics in the fermented beverage, including increased levels oforganic acids and/or reduced levels of acetic acid.

Non-limiting examples of fermented beverages include kombucha, waterkefir, flavored water product, jun, soda, seltzer, sparkling water, gutshot, functional beverage, fermented fruit juice, fermented vegetablejuice, probiotic beverages, ginger beer, and fermented sports drinks. Insome embodiments, the fermented beverage is kombucha.

The fermented beverages described herein are fermented to completion,meaning the fermentation process used to produce the beverages isperformed under conditions and for a time period sufficient to bring thelevel of one or more components below (or in some instances above) acertain level. For example, in some instances, fermented to completionmeans that the fermentation process is performed under conditions andfor a time period sufficient to bring the sugar level in the fermentedbeverage product to below a threshold level. In some embodiments,fermented to completion means that the fermentation process is performedunder conditions and for a time period sufficient to bring the level ofalcohol (ethanol) to below a threshold level. In some instances,fermented to completion means that the fermentation process is performedunder conditions and for a time period sufficient to bring the sugarlevel in the fermented beverage product to below a threshold level andto bring the level of alcohol (ethanol) to below a threshold level. Insome embodiments, the final fermented beverage contains a level of sugarthat is less than the level of sugar, for example, in a fermentedbeverage that is produced using other microbial communities. In someembodiments, the fermented beverage contains a level of sugar that isless than 20.0 g/L, less than 19.0 g/L, less than 18.0 g/L, less than17.0 g/L, less than 16.0 g/L, less than 15.0 g/L, less than 14.0 g/L,less than 13.0 g/L, less than 12.0 g/L, less than 11.0 g/L, less than10.0 g/L, less than 9.0 g/L, less than 8.0 g/L, less than 7.0 g/L, lessthan 6.0 g/L, less than 5.0 g/L, less than 4.0 g/L, less than 3.0 g/L,less than 2.0 g/L, less than 1.0 g/L, less than 0.9 g/L, less than 0.8g/L, less than 0.07 g/L, less than 0.6 g/L, less than 0.5 g/L, less than0.4 g/L, less than 0.3 g/L, less than 0.2 g/L, less than 0.1 g/L. Insome embodiments, the fermented beverage contains a level of sugar thatis not detectable (below the limits of detection). In some embodiments,the fermented product does not contain sugar, e.g., approximately 0 g/L.

In some embodiments, the fermented beverages described herein areconsidered “zero sugar” or “zero calorie” fermented beverages. Accordingto the U.S. Food and Drug Administration, a food or beverage productmust contain less than 0.5 grams sugar per serving (e.g., 8 fluid ounceserving) to be a “zero sugar” food or beverage product and less than 5calories per serving to be a “zero calorie” food or beverage product.

In some embodiments, the fermented beverage is considered a “low sugar”fermented beverage. As used herein, the term “low sugar” refers to alevel of sugar that is less than 5 grams per 8 fluid ounce serving. Insome embodiments, the fermented beverages described herein contain lessthan 10.0 g, less than 9.0 g, 0.5 g, less than 0.4 g, less than 0.3 g,less than 0.3 g, less than 0.2 g, less than 0.1 g, less than 0.09 g,less than 0.08 g, less than 0.07 g, less than 0.06 g, less than 0.05 g,less than 0.04 g, less than 0.03 g, less than 0.02 g, less than 0.01 g,or lower per 8 fluid ounce serving (approximately 237 milliliters).

The level of sugar (sugar content) in a product, such as a fermentedbeverage, may be assessed using methods known in the art, for examplegas chromatography or enzymatic assays.

Described herein, fermentation processes involve the conversion ofcarbohydrates into alcohol and carbon dioxide, and possibly theconversion of the alcohol to organic acids. By way of an example, duringfermentation of kombucha using microbial communities containing yeaststrains in addition to bacterial strains, the fermentation processincludes two steps: conversion of sugars to alcohol by the yeast strainsand subsequently conversion of alcohol to organic acids by the yeast andbacterial strains. However, during kombucha fermentation, the secondstep of fermentation is not complete, resulting in residual alcohollevels up to around 3% alcohol by volume (i.e., 3% of the total volumeis alcohol) reported for some home-brewed kombuchas. See, e.g.,brewdrkombucha.com, Blog post, Sep. 28, 2018: “Alcohol in Kombucha: Whatyou need to know.”

The level of alcohol in a fermented beverage is typically reported asalcohol by volume (also referred to as “ABV,” “abv,” or “alc/vol”),which is presented as a volume percent. ABV is typically used as ameasure of the amount of ethanol in beverage products and is calculatedas the number of milliliters of ethanol per 100 mL of beverage productat 20° C. In some embodiments, the fermented beverage contains a levelof alcohol (ethanol) that is less than 3%, less than 2%, less than 1%,less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, lessthan 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than0.1% v/v or lower. In some embodiments, the fermented beverage isnon-alcoholic (e.g., has an alcohol by volume less than 0.5%).

The amount of alcohol (e.g., ethanol) in a beverage product can bemeasured by any means known in the art, such as using the specificgravity or an enzymatic assay. The specific gravity may be measuredprior to and after a fermentation process and the change in specificgravity indicates the amount of alcohol produced during fermentation.Alternatively or in addition, enzymatic assays for alcohol (e.g.,ethanol) or near infrared alcohol measurements (e.g., “Alcolyzer”) maybe used to determine the amount of alcohol in a beverage product.

During the fermentation process, microorganisms metabolize carbohydratesinto organic acids, which may contribute to the flavor and/or aromaticprofile of a fermented beverage. The presence of organic acids in thefermented product may provide desired sensory qualities, such as desiredflavors, tastes, and aromas, as well as health benefits. In particular,glucuronic acid has been attributed with providing numerous healthbenefits, including as a precursor to the synthesis of vitamin C and asa substrate for production of glucosamine, which is used in theprophylaxis of arthritis. See, e.g., Nguyen et al. LWT—Food Science andTechnology (2015) 64, 1149-1155; Yavari et al. Australian Journal ofBasic and Applied Sciences (2011) 5(11), 1788-1794.

Examples of organic acids produced during fermentation include, withoutlimitation, acetic acid, glucuronic, ketogluconic acid, gluconic acid,lactic acid, malic acid, citric acid, tartaric acid, folic acid, malonicacid, oxalic acid, succinic acid, pyruvic acid, and usnic acid. See,e.g., Neffe-Skocinska et al. CyTA—Journal of Food (2017) 15:4, 601-607.

Kombuchas produced using conventional methods may be characterized byrelatively high levels of acetic acid, which impart an undesirablevinegar flavor. As used herein, high levels of acetic acid refer tolevels of acetic acid that are about 10 g/L or higher. In someembodiments, the fermented beverages described herein contain a reducedlevel of acetic acid (e.g., less than about 10 g/L), for example ascompared to fermented beverages produced using other microbialcommunities, such as microbial communities containing yeast strains. Insome embodiments, the fermented beverages described herein contain lessthan 10 g/L acetic acid. In some embodiments, the fermented beveragesdescribed herein contain a level of acetic acid that is less than 10.0g/L, 9.0 g/L, 8.0 g/L, 7.0 g/L, 6.0 g/L, 5.0 g/L, 4.0 g/L, 3.0 g/L, 2.0g/L, 1.0 g/L, 0.9 g/L, 0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3g/L, 0.2 g/L, 0.1 g/L, 0.09 g/L, 0.08 g/L, 0.07 g/L, 0.06 g/L, 0.05 g/L,0.04 g/L, 0.03 g/L, 0.02 g/L, 0.01 g/L or lower. In some embodiments,the fermented beverages described herein contain a level of acetic acidthat is between 0.1-0.5 g/L.

In some embodiments, the fermented beverages described herein containincreased levels of organic acids that are not acetic acid, for exampleas compared to the level of organic acids present in fermented beveragesproduced using other microbial communities, such as microbialcommunities containing yeast strains. In some embodiments, the fermentedbeverages described herein contain more than 10.0 g/L organic acids thatare not acetic acid. In some embodiments, the fermented beveragesdescribed herein contain more than 10.0 g/L lactic acid, gluconic acid,ketogluconic acid, or a combination thereof. In some embodiments, thefermented beverages described herein contain a level of organic acid(s)that is more than 1.0 g/L, 2.0 g/L, 3.0 g/L, 4.0 g/L, 5.0 g/L, 6.0 g/L,7.0 g/L, 8.0 g/L, 9.0 g/L, 10.0 g/L, 11.0 g/L, 12.0 g/L, 13.0 g/L, 14.0g/L, 15.0 g/L, 16.0 g/L, 17.0 g/L, 18.0 g/L, 19.0 g/L, 20.0 g/L, 21.0g/L, 22.0 g/L, 23.0 g/L, 24.0 g/L, 25.0 g/L, 26.0 g/L, 27.0 g/L, 28.0g/L, 29.0 g/L, 30.0 g/L, 31.0 g/L, 32 g/L, 33 g/L, 34 g/L, 35 g/L, 36g/L, 37 g/L, 38 g/L, 39 g/L, or up to 40 g/L.

In some embodiments, the fermented beverages described herein contain alevel of gluconic acid that is between 0.5 g/L-2.0 g/L. In someembodiments, the fermented beverages described herein contain a level ofgluconic acid that is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 g/L.

In some embodiments, the fermented beverages described herein contain alevel of lactic acid that is between 0.02 g/L-2.0 g/L. In someembodiments, the fermented beverages described herein contain a level oflactic acid that is about 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.6 g/L,0.7 g/L, 0.8 g/L, 0.9 g/L, 1.0 g/L, 1.1 g/L, 1.2 g/L, 1.3 g/L, 1.4, g/L1.5 g/L, 1.6 g/L, 1.7 g/L, 1.8 g/L, 1.9 g/L, or 2.0 g/L.

In some embodiments, the pH of the fermented beverage is acidic, i.e.,less than pH 6.0. For a shelf-stable acidified beverage, the U.S. Foodand Drug Administration Food Code requires that the final pH of thebeverage be less than pH 4.6. In some embodiments, the pH of thefermented beverage is less than about pH 6, 5.9, 5.8, 5.7, 5.6, 5.5,5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.6, 4.5, 4.4, 4.3, 4.2,4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8,2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0 or lower. In some embodiments,the pH of the fermented beverage is less than about 4.6. In someembodiments, the pH of the fermented beverage is less than about 3.5. Insome embodiments, the pH of the fermented beverage between 2.9-3.1.

As described herein, many fermented beverages, such as kombucha, must bestored and distributed using refrigerated temperatures (i.e., coldsupply chain), to protect integrity of the product; and/or prevent orreduce continued fermentation, including increase in alcohol content;and/or prevent or reduce microbial overgrowth. Recommended temperaturesfor storing and distributing kombucha products, for example, areconsidered to be between 34-40° F. (1.1-4.4° C.) by Kombucha BrewersInternational. Maintaining refrigerated temperatures during storage anddistribution is substantially more expensive relative to roomtemperature (ambient temperature), non-refrigerated conditions.Moreover, failure to maintain appropriately cold temperatures for theprior art kombucha products may result in an inferior fermented product,overgrowth of microbial strains including contaminants, and explosion ofbottles containing the kombucha product.

In contrast, the fermented beverages described herein have increasedshelf-stability. As used herein, the term “shelf-stable” refers tostability and product integrity when a product is maintained at ambienttemperatures (about 20°-22° C.) for an extended period of time. In someembodiments, the fermented beverage is stable at ambient temperature(i.e., between about 20°-22° C.) for at least or about 1 week, at leastor about 2 weeks, at least or about 3 weeks, at least or about 4 weeks,at least or about 5 weeks, at least or about 6 weeks, at least or about7 weeks, at least or about 8 weeks, at least or about one month, atleast or about two months, at least or about three months, at least orabout four months, at least or about five months, at least or about sixmonths, at least or about seven months, at least or about eight months,at least or about nine months, at least or about ten months, at least orabout eleven months, at least or about twelve months, at least or aboutone year, at least or about two years, at least or about three years, atleast or about four years, at least or about five years, or longer.

In some embodiments, the fermented beverages described herein may alsohave increased stability or product integrity at an elevatedtemperature, e.g., above about 23° C. In some embodiments, the fermentedbeverages described herein have increased stability or product integrityat a temperature higher than or about 23° C., higher than or about 24°C., higher than or about 25° C., higher than or about 26° C., higherthan or about 27° C., higher than or about 28° C., about 29° C., higherthan or about 30° C., higher than or about 31° C., higher than or about32° C., higher than or about 33° C., higher than or about 34° C., higherthan or about 35° C., higher than or about 36° C., higher than or about37° C., higher than or about 38° C., higher than or about 39° C., higherthan or about 40° C. or higher.

In some embodiments, the fermented beverage is stable at an elevatedtemperature, e.g., above or about 23° C., for at least or about 1 week,at least or about 2 weeks, at least or about 3 weeks, at least or about4 weeks, at least or about 5 weeks, at least or about 6 weeks, at leastor about 7 weeks, at least or about 8 weeks, at least or about onemonth, at least or about two months, at least or about three months, atleast or about four months, at least or about five months, at least orabout six months, at least or about seven months, at least or abouteight months, at least or about nine months, at least or about tenmonths, at least or about eleven months, at least or about twelvemonths, at least or about one year, at least or about two years, atleast or about three years, at least or about four years, at least orabout five years, or longer.

Determining whether a fermented beverage is stable or the productintegrity is intact will be evident to one of ordinary skill in the art,and may involve using methods known in the art. For example, in someembodiments, the stability of the fermented beverage under particularconditions involves evaluating, for example, flavor profile, colorprofile, amounts of CFUs of microbial strains, alcohol content, pH,and/or detection of contaminants. In general, a fermented beverage isconsidered to be “stable” if one or more properties (e.g., CFU of themicrobial strains, sugar content, alcohol content, flavor profile, pH,etc) of the beverages does not substantially change under particularconditions.

In some embodiments, a fermented product is considered shelf-stable ifthe number of (viable) microbial cells does not change by more than 90%over a period of at least one week, two weeks, three weeks, four weeks,five weeks, six weeks, or longer at ambient temperature. In someembodiments, a fermented product is considered shelf-stable if the pH ofthe fermented product does not change by more than 10% over a period ofat least one week, two weeks, three weeks, four weeks, five weeks, sixweeks, or longer at ambient temperature. In some embodiments, afermented product is considered shelf-stable if the product flavorprofile does not substantially or detectably change over a period of atleast one week, two weeks, three weeks, four weeks, five weeks, sixweeks, or longer at ambient temperature.

A fermented beverage may be considered to be stable in regard to thebiomass if there is less than 99% decrease (2-logs) in the biomass inthe fermented beverage over 60 days. In some embodiments, the fermentedbeverage has a biomass (CFU/mL) that decreases by less than 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%. 99%. 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.8%, 99.9% or higherover a period of 60 days, for example stored at room temperature. Insome embodiments, the fermented beverage has a biomass (CFU/mL) thatdecreases by less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%. 99%. 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.8%, 99.9% or higher over a period of 60 days as compared tothe biomass of the fermented beverage at a prior time point (e.g., atthe end of fermentation), another microbial community, or individualmicrobial strains.

In some embodiments, the fermented beverage contains a microbialcommunity having a biomass (CFU/mL) that decreases by less than1,000-fold (3 logs), 100-fold (2 logs), or less than 10-fold (1 log)over a period of 60 days, for example stored at room temperature. Insome embodiments, the fermented beverage contains a microbial communityhaving a biomass (CFU/mL) that decreases by less than 1,000-fold (3logs), 100-fold (2 logs), or less than 10-fold (1 log) over a period of60 days, for example as compared to the biomass of the fermentedbeverage at a prior time point (e.g., at the end of fermentation),another microbial community, or individual microbial strains.

In some embodiments, the fermented beverages comprise about 10, about10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸,about 10⁹, about 10¹⁰, about 10¹¹, about 10¹², about 10¹³ or more ofeach of the microbial strains per 8 fluid ounce serving (approximately237 mL). In some embodiments, the fermented beverages comprise at leastor about 10, at least or about 10², at least or about 10³, at least orabout 10⁴, at least or about 10⁵, at least or about 10⁶, at least orabout 10⁷, at least or about 10⁸, at least or about 10⁹, at least orabout 10¹⁰, at least or about 10¹¹, at least or about 10¹², at least orabout 10¹³ or more of each of the microbial strains per 8 fluid ounceserving (approximately 237 mL).

In some embodiments, the fermented beverages comprise about 10, about10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸,about 10⁹, about 10¹⁰, about 10¹¹, about 10¹², about 10¹³ or more ofeach of the microbial strains per milliliter of fermented beverage. Insome embodiments, the fermented beverages comprise at least or about 10,at least or about 10², at least or about 10³, at least or about 10⁴, atleast or about 10⁵, at least or about 10⁶, at least or about 10⁷, atleast or about 10⁸, at least or about 10⁹, at least or about 10¹⁰, atleast or about 10¹¹, at least or about 10¹², at least or about 10¹³ ormore of each of the microbial strains per milliliter of fermentedbeverage.

In some embodiments, the fermented beverage contains between about 10and about 10¹³, between about 10² and about 10¹³, between about 10³ andabout 10¹³, between about 10⁴ and about 10¹³, between about 10⁵ andabout 10¹³, between about 10⁶ and about 10¹³, between about 10⁷ andabout 10¹³, between about 10⁸ and about 10¹³, between about 10⁹ andabout 10¹³, between about 10¹⁰ and about 10¹³, between about 10¹¹ andabout 10¹³, between about 10¹² and about 10¹³, between about 10 andabout 10¹², between about 10² and about 10¹², between about 10³ andabout 10¹², between about 10⁴ and about 10¹², between about 10⁵ andabout 10¹², between about 10⁶ and about 10¹², between about 10⁷ andabout 10¹², between about 10⁸ and about 10¹², between about 10⁹ andabout 10¹², between about 10¹⁰ and about 10¹², between about 10¹¹ andabout 10¹², between about 10 and about 10¹¹, between about 10² and about10¹¹, between about 10³ and about 10¹³, between about 10⁴ and about10¹³, between about 10⁵ and about 10¹³, between about 10⁶ and about10¹³, between about 10⁷ and about 10¹¹, between about 10⁸ and about10¹¹, between about 10⁹ and about 10¹¹, between about 10¹⁰ and about10¹¹, between about 10 and about 10¹⁰, between about 10² and about 10¹⁰,between about 10³ and about 10¹⁰, between about 10⁴ and about 10¹⁰,between about 10⁵ and about 10¹⁰, between about 10⁶ and about 10¹⁰,between about 10⁷ and about 10¹⁰, between about 10⁸ and about 10¹⁰,between about 10⁹ and about 10¹⁰, between about 10 and about 10⁹,between about 10² and about 10⁹, between about 10³ and about 10⁹,between about 10⁴ and about 10⁹, between about 10⁵ and about 10⁹,between about 10⁶ and about 10⁹, between about 10⁷ and about 10⁹,between about 10⁸ and about 10⁹, between about 10 and about 10⁸, betweenabout 10² and about 10⁸, between about 10³ and about 10⁸, between about10⁴ and about 10⁸, between about 10⁵ and about 10⁸, between about 10⁶and about 10⁸, between about 10⁷ and about 10⁸, between about 10 andabout 10⁷, between about 10² and about 10⁷, between about 10³ and about10⁷, between about 10⁴ and about 10⁷, between about 10⁵ and about 10⁷,between about 10⁶ and about 10⁷, between about 10 and about 10⁶, betweenabout 10² and about 10⁶, between about 10³ and about 10⁶, between about10⁴ and about 10⁶, between about 10⁵ and about 10⁶, between about 10 andabout 10⁵, between about 10² and about 10⁵, between about 10³ and about10⁵, between about 10⁴ and about 10⁵, between about 10 and about 10⁴,between about 10² and about 10⁴, between about 10³ and about 10⁴,between about 10 and about 10³, between about 10² and about 10³, orbetween about 10 and about 10² of each of the microbial strains per 8fluid ounce serving. In some embodiments, the fermented beveragecontains between 10⁷ and 10⁹ of each of the microbial strains per 8fluid ounce serving. Each of the microbial strains of the symbioticmicrobial community may be present in the same amount or in differentamounts.

In some embodiments, the fermented beverage contains between about 10and about 10¹³, between about 10² and about 10¹³, between about 10³ andabout 10¹³, between about 10⁴ and about 10¹³, between about 10⁵ andabout 10¹³, between about 10⁶ and about 10¹³, between about 10⁷ andabout 10¹³, between about 10⁸ and about 10¹³, between about 10⁹ andabout 10¹³, between about 10¹⁰ and about 10¹³, between about 10¹¹ andabout 10¹³, between about 10¹² and about 10¹³, between about 10 andabout 10¹², between about 10² and about 10¹², between about 10³ andabout 10¹², between about 10⁴ and about 10¹², between about 10⁵ andabout 10¹², between about 10⁶ and about 10¹², between about 10⁷ andabout 10¹², between about 10⁸ and about 10¹², between about 10⁹ andabout 10¹², between about 10¹⁰ and about 10¹², between about 10¹¹ andabout 10¹², between about 10 and about 10¹¹, between about 10² and about10¹¹, between about 10³ and about 10¹³, between about 10⁴ and about10¹³, between about 10⁵ and about 10¹³, between about 10⁶ and about10¹³, between about 10⁷ and about 10¹¹, between about 10⁸ and about10¹¹, between about 10⁹ and about 10¹¹, between about 10¹⁰ and about10¹¹, between about 10 and about 10¹⁰, between about 10² and about 10¹⁰,between about 10³ and about 10¹⁰, between about 10⁴ and about 10¹⁰,between about 10⁵ and about 10¹⁰, between about 10⁶ and about 10¹⁰,between about 10⁷ and about 10¹⁰, between about 10⁸ and about 10¹⁰,between about 10⁹ and about 10¹⁰, between about 10 and about 10⁹,between about 10² and about 10⁹, between about 10³ and about 10⁹,between about 10⁴ and about 10⁹, between about 10⁵ and about 10⁹,between about 10⁶ and about 10⁹, between about 10⁷ and about 10⁹,between about 10⁸ and about 10⁹, between about 10 and about 10⁸, betweenabout 10² and about 10⁸, between about 10³ and about 10⁸, between about10⁴ and about 10⁸, between about 10⁵ and about 10⁸, between about 10⁶and about 10⁸, between about 10⁷ and about 10⁸, between about 10 andabout 10⁷, between about 10² and about 10⁷, between about 10³ and about10⁷, between about 10⁴ and about 10⁷, between about 10⁵ and about 10⁷,between about 10⁶ and about 10⁷, between about 10 and about 10⁶, betweenabout 10² and about 10⁶, between about 10³ and about 10⁶, between about10⁴ and about 10⁶, between about 10⁵ and about 10⁶, between about 10 andabout 10⁵, between about 10² and about 10⁵, between about 10³ and about10⁵, between about 10⁴ and about 10⁵, between about 10 and about 10⁴,between about 10² and about 10⁴, between about 10³ and about 10⁴,between about 10 and about 10³, between about 10² and about 10³, orbetween about 10 and about 10² of each of the microbial strains permilliliter of fermented beverage. In some embodiments, the fermentedbeverage contains between 10⁵ and 10⁶ of each of the microbial strainsper milliliter of fermented beverage. Each of the microbial strains ofthe symbiotic microbial community may be present in the same amount orin different amounts.

In some embodiments, the fermented beverages comprise about 10, about10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸,about 10⁹, about 10¹⁰, about 10¹¹, about 10¹², about 10¹³ or more totalmicrobial cells (CFU) of all microbial strains per 8 fluid ounce serving(approximately 237 mL). In some embodiments, the fermented beveragescomprise at least 10, at least 10², at least 10³, at least 10⁴, at least10⁵, at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹, at least10¹⁰, at least 10¹¹, at least 10¹², at least 10¹³ or more of totalmicrobial cells (CFU) of all microbial strains per 8 fluid ounce serving(approximately 237 mL)).

In some embodiments, the fermented beverages comprise about 10, about10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸,about 10⁹, about 10¹⁰, about 10¹¹, about 10¹², about 10¹³ or more totalmicrobial cells (CFU) of all microbial strains per milliliter fermentedbeverage. In some embodiments, the fermented beverages comprise at least10, at least 10², at least 10³, at least 10⁴, at least 10⁵, at least10⁶, at least 10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰, at least10¹¹, at least 10¹², at least 10¹³ or more of total microbial cells(CFU) of all microbial strains per milliliter fermented beverage.

In some embodiments, the fermented beverage contains between about 10and about 10¹³, between about 10² and about 10¹³, between about 10³ andabout 10¹³, between about 10⁴ and about 10¹³, between about 10⁵ andabout 10¹³, between about 10⁶ and about 10¹³, between about 10⁷ andabout 10¹³, between about 10⁸ and about 10¹³, between about 10⁹ andabout 10¹³, between about 10¹⁰ and about 10¹³, between about 10¹¹ andabout 10¹³, between about 10¹² and about 10¹³, between about 10 andabout 10¹², between about 10² and about 10¹², between about 10³ andabout 10¹², between about 10⁴ and about 10¹², between about 10⁵ andabout 10¹², between about 10⁶ and about 10¹², between about 10⁷ andabout 10¹², between about 10⁸ and about 10¹², between about 10⁹ andabout 10¹², between about 10¹⁰ and about 10¹², between about 10¹¹ andabout 10¹², between about 10 and about 10¹¹, between about 10² and about10¹¹, between about 10³ and about 10¹³, between about 10⁴ and about10¹³, between about 10⁵ and about 10¹³, between about 10⁶ and about10¹³, between about 10⁷ and about 10¹¹, between about 10⁸ and about10¹¹, between about 10⁹ and about 10¹¹, between about 10¹⁰ and about10¹¹, between about 10 and about 10¹⁰, between about 10² and about 10¹⁰,between about 10³ and about 10¹⁰, between about 10⁴ and about 10¹⁰,between about 10⁵ and about 10¹⁰, between about 10⁶ and about 10¹⁰,between about 10⁷ and about 10¹⁰, between about 10⁸ and about 10¹⁰,between about 10⁹ and about 10¹⁰, between about 10 and about 10⁹,between about 10² and about 10⁹, between about 10³ and about 10⁹,between about 10⁴ and about 10⁹, between about 10⁵ and about 10⁹,between about 10⁶ and about 10⁹, between about 10⁷ and about 10⁹,between about 10⁸ and about 10⁹, between about 10 and about 10⁸, betweenabout 10² and about 10⁸, between about 10³ and about 10⁸, between about10⁴ and about 10⁸, between about 10⁵ and about 10⁸, between about 10⁶and about 10⁸, between about 10⁷ and about 10⁸, between about 10 andabout 10⁷, between about 10² and about 10⁷, between about 10³ and about10⁷, between about 10⁴ and about 10⁷, between about 10⁵ and about 10⁷,between about 10⁶ and about 10⁷, between about 10 and about 10⁶, betweenabout 10² and about 10⁶, between about 10³ and about 10⁶, between about10⁴ and about 10⁶, between about 10⁵ and about 10⁶, between about 10 andabout 10⁵, between about 10² and about 10⁵, between about 10³ and about10⁵, between about 10⁴ and about 10⁵, between about 10 and about 10⁴,between about 10² and about 10⁴, between about 10³ and about 10⁴,between about 10 and about 10³, between about 10² and about 10³, orbetween about 10 and about 10² total microbial cells (e.g., CFU) per 8fluid ounce serving. In some embodiments, the fermented beveragecontains between 10⁷ and 10⁹ microbial cells (e.g., CFU) total per 8fluid ounce serving.

In some embodiments, the fermented beverage contains between about 10and about 10¹³, between about 10² and about 10¹³, between about 10³ andabout 10¹³, between about 10⁴ and about 10¹³, between about 10⁵ andabout 10¹³, between about 10⁶ and about 10¹³, between about 10⁷ andabout 10¹³, between about 10⁸ and about 10¹³, between about 10⁹ andabout 10¹³, between about 10¹⁰ and about 10¹³, between about 10¹¹ andabout 10¹³, between about 10¹² and about 10¹³, between about 10 andabout 10¹², between about 10² and about 10¹², between about 10³ andabout 10¹², between about 10⁴ and about 10¹², between about 10⁵ andabout 10¹², between about 10⁶ and about 10¹², between about 10⁷ andabout 10¹², between about 10⁸ and about 10¹², between about 10⁹ andabout 10¹², between about 10¹⁰ and about 10¹², between about 10¹¹ andabout 10¹², between about 10 and about 10¹¹, between about 10² and about10¹¹, between about 10³ and about 10¹³, between about 10⁴ and about10¹³, between about 10⁵ and about 10¹³, between about 10⁶ and about10¹³, between about 10⁷ and about 10¹¹, between about 10⁸ and about10¹¹, between about 10⁹ and about 10¹¹, between about 10¹⁰ and about10¹¹, between about 10 and about 10¹⁰, between about 10² and about 10¹⁰,between about 10³ and about 10¹⁰, between about 10⁴ and about 10¹⁰,between about 10⁵ and about 10¹⁰, between about 10⁶ and about 10¹⁰,between about 10⁷ and about 10¹⁰, between about 10⁸ and about 10¹⁰,between about 10⁹ and about 10¹⁰, between about 10 and about 10⁹,between about 10² and about 10⁹, between about 10³ and about 10⁹,between about 10⁴ and about 10⁹, between about 10⁵ and about 10⁹,between about 10⁶ and about 10⁹, between about 10⁷ and about 10⁹,between about 10⁸ and about 10⁹, between about 10 and about 10⁸, betweenabout 10² and about 10⁸, between about 10³ and about 10⁸, between about10⁴ and about 10⁸, between about 10⁵ and about 10⁸, between about 10⁶and about 10⁸, between about 10⁷ and about 10⁸, between about 10 andabout 10⁷, between about 10² and about 10⁷, between about 10³ and about10⁷, between about 10⁴ and about 10⁷, between about 10⁵ and about 10⁷,between about 10⁶ and about 10⁷, between about 10 and about 10⁶, betweenabout 10² and about 10⁶, between about 10³ and about 10⁶, between about10⁴ and about 10⁶, between about 10⁵ and about 10⁶, between about 10 andabout 10⁵, between about 10² and about 10⁵, between about 10³ and about10⁵, between about 10⁴ and about 10⁵, between about 10 and about 10⁴,between about 10² and about 10⁴, between about 10³ and about 10⁴,between about 10 and about 10³, between about 10² and about 10³, orbetween about 10 and about 10² total microbial cells (e.g., CFU) permilliliter of fermented beverage. In some embodiments, the fermentedbeverage contains between 10⁵ and 10⁶ total microbial cells (e.g., CFU)per milliliter of fermented beverage. As discussed above, a microbialstrain may be present in the same amount or a different amount ascompared to another microbial strain.

The fermented beverages described herein may include one or moreadditional components that may contribute flavoring, aromatic,preservation, colorant, caffeine, sweetness, nutritive and/or functionalproperties to the beverage. In some embodiments, the fermented beveragemay include one or more sweetening agents, such as natural sweetenersand artificial sweeteners. In some embodiments, the fermented beverageincludes monk fruit sweetener. In some embodiments, the fermentedbeverage includes one or more sweet proteins. In some embodiments, thefermented beverage includes sucralose, tagatose, sugar alcohols(polyols, such as mannitol, sorbitol, xylito), aspartame, saccharin,acesulfame K, and/or stevia.

In some embodiments, the fermented beverage contains one or moreadditional flavoring components. Examples of additional flavoringcomponents (natural, naturally derived, or synthetic) include, withoutlimitation, fruit flavoring, including fruit extract, fruit juice, fruitpeel, and fruit puree, such as apple, pear, blackberry, grape,nectarines, watermelon, banana pineapple, cherry, cranberry, lemon,lime, orange, citrus, grapefruit, cranberry, blueberry, quince,raspberry, strawberry, fig, apricot, plum, blackberry, pomelo, melon,mango, kiwi; vegetable flavoring, including vegetable extract, vegetablejuice, and vegetable puree, such as carrot, celery, lettuce, wheatgrass,kale, broccoli, beans, cauliflower, cucumbers, squash, turnips,potatoes, yams, beets; herbs and spices, such as ginger, black pepper,cardamon, cinnamon, clove, nutmeg, chiles, mint, turmeric.

In some embodiments, the fermented beverage contains one or moreadditional nutritive components. Examples of nutritive components(natural, naturally derived, or synthetic) include, without limitationvitamins, minerals, and antioxidants, such as vitamin B complex,ascorbic acid (vitamin C), thiamine (vitamin B1), riboflavin (vitaminB2), niacin (vitamin B3), D-calcium pantothenate (vitamin B5), vitaminB6, vitamin B12, calcium, iron, vitamin D, vitamin E, vitamin A, vitaminK, magnesium, potassium, pantothenic acid, folic acid, zinc, andmagnesium.

A fermented beverage that contains one or more nutritive components maybe referred to as “fortified,” such as vitamin and/or mineral fortified.

In some embodiments, the fermented beverages described herein furthercomprise tea or tea extract, such as non-fermented tea, semi-fermentedtea, or fermented tea and extracts thereof. Examples of tea that may beused in the fermented beverages described herein include, withoutlimitation, white tea, yellow tea, green tea, such as sencha, bancha,mochi tea, kettle tea, stem tea, stick tea, and bud tea: oolong tea,such as iron kannon, color type, golden katsura, and martial arts tea;black tea, Darjeeling, Assam, Ceylon, Sri Lanka, Keemun, Yunnan, Bai LinGong Fu; Nilgiri; herbal tea, and tea blends, such as Earl Grey, Englishbreakfast, English afternoon, Irish breakfast, and masala chai. As willbe evident to one of ordinary skill in the art, soluble compounds of tealeaves are typically extracted by hot water and/or steam and may beadded to the fermented beverage.

Alternatively or in addition, any of the fermented beverages describedherein may contain one or more functional components. As used herein,the term “functional component” refers to a component that may imparthealth benefits to the consumer other than basic nutritional components,such as promote hydration; promote healthy skin, nails, hair; increaseenergy; promote relaxation; enhance mood; improve digestion; promoteimmunity. Examples of functional components that may be used in thefermented beverages described herein include, without limitation,potassium, bamboo, biotin, silica, lavender, vanilla, passionflower,orange, rose, cucumber, lemon, strawberry, L-theanine, ginger,pineapple, black pepper, and turmeric.

Any of the fermented beverages described herein may further comprise oneor more probiotic microbial strain, such as Bacillus coagulans, that maybe added, for example, to the final product.

Methods of Production

Aspects of the present disclosure relate to methods of producing afermented beverage using any of the symbiotic microbial communitiesdescribed herein. In general, fermentation processes harness the abilityof microorganisms to convert sugars into alcohol and organic acids. Inthe context of food and beverage production, fermentation broadly refersto any process in which the activity of microorganisms brings about adesirable change to a food product or beverage. The conditions forfermentation and the carrying out of a fermentation are referred toherein as a “fermentation process.”

The methods of producing a fermented beverage described herein, such askombucha, involve providing a medium comprising at least one fermentablesugar at an initial sugar level, adding any of the symbiotic microbialcommunities described herein to the medium to produce a culture, andsubjecting the culture to suitable conditions for a period of timesufficient to ferment the culture (i.e., a fermentation process),thereby producing a fermented beverage. The microbial communities may bein the form of a starter culture containing each of the microbialstrains of the community or in the form of multiple starter cultureseach of which contains one microbial strain that when combined form thesymbiotic microbial community. Starter cultures may be in any suitableform for adding to a medium, such as liquid, frozen, refrigerated,freeze dried, or lyophilized.

As used herein, the term “medium” refers to a composition that isconducive to fermentation, meaning a composition that does not inhibitfermentation. The medium may contain one or more fermentable sugar ormay be supplemented with one or more fermentable sugar. In someembodiments, the medium is tea, water, juice, or extract.

The term “fermentable sugar,” as used herein, refers to a carbohydratethat may be converted into an alcohol and carbon dioxide and ultimatelyto organic acids by microbial strains or a community thereof, such asany of the symbiotic microbial communities described herein. Examples offermentable sugars include, without limitation, glucose, fructose,lactose, sucrose, maltose, and maltotriose.

In some embodiments, the method further involves adding the fermentablesugar to the medium. In some embodiments, the fermentable sugar is addedto the medium prior to or during the fermentation process as a sugarsource for the microbial strains. The sugar source for use in thefermented beverages and methods described herein may depend, forexample, on the type of fermented beverage product and the fermentablesugar. Examples of sugar sources include, without limitation, fruitjuice, fruit extract, vegetable juice, vegetable extract, honey,purified sugar such as cane sugar, palm sugar, beet sugar, maple syrup,brown sugar, molasses, agave nectar, honey, date syrup, date paste, datesugar, coconut sugar, monk fruit sweetener, and coconut water. In someembodiments, the sugar source is an artificial sugar source.

In some embodiments, the medium may comprise one or more additionalflavoring component, that may be added prior to fermentation (e.g.,teas, spices, natural flavorings, herbs). Examples of additionalflavoring components (natural, naturally derived, or synthetic) include,without limitation, fruit flavoring, including fruit extract, fruitjuice, fruit peel, and fruit puree, such as apple, pear, blackberry,grape, nectarines, watermelon, banana pineapple, cherry, cranberry,lemon, lime, orange, citrus, grapefruit, cranberry, blueberry, quince,raspberry, strawberry, fig, apricot, plum, blackberry, pomelo, melon,mango, kiwi; vegetable flavoring, including vegetable extract, vegetablejuice, and vegetable puree, such as carrot, celery, lettuce, wheatgrass,kale, broccoli, beans, cauliflower, cucumbers, squash, turnips,potatoes, yams, beets, ginger, black pepper, cardamon, cinnamon, clove,nutmeg, chiles, mint, and turmeric. In some embodiments, the mediumfurther comprises ginger or extracts derived or obtained from ginger.

As described herein, the medium comprises a fermentable sugar at aninitial sugar level. During the fermentation process the fermentablesugar is converted to alcohol and organic acids, thereby reducing thesugar level in the medium to a final sugar level (content) in thefermented beverage that is lower than the initial sugar level. In someembodiments, the initial sugar level in the medium is about 2.5 g/L-20g/L. In some embodiments, the initial sugar level in the medium is about1.0 g/L, about 1.5 g/L, about 2.0 g/L, about 2.5 g/L, about 3.0 g/L,about 3.5 g/L, about 4.0 g/L, about 4.5 g/L, about 5.0 g/L, about 5.5g/L, about 6.0 g/L, about 6.5 g/L, about 7.0 g/L, about 7.5 g/L, about8.0 g/L, about 8.5 g/L, about 9.0 g/L, about 9.5 g/L, about 10 g/L,about 11 g/L, about 12 g/L, about 13 g/L, about 14 g/L, about 15 g/L,about 16 g/L, about 17 g/L, about 18 g/L, about 19 g/L, about 20 g/L.

In some embodiments, the initial pH of the medium is at or below 4.6. Insome embodiments, the pH of the medium may be adjusted to reach aninitial pH at or below 4.6. Suitable methods of adjusting the pH of amedium will be evident to one of ordinary skill in the art and mayinvolve adding an acid, such as lemon juice, to the medium.

In some embodiments, fermentation conditions for use in the methodsdescribed herein that produce a fermented beverage may be carried outfor about 1 day to about 60 days. In some embodiments, the fermentationprocess is performed for about 1 day, about 2 days, about 3 days, about4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, about 11 days, about 12 days, about 13 days, about14 days, about 15 days, about 16 days, about 17 days, about 18 days,about 19 days, about 20 days, about 21 days, about 22 days, about 23days, about 24 days, about 25 days, about 26 days, about 27 days, about28 days, about 29 days, about 30 days, about 35 days, about 40 days,about 45 days, about 50 days, about 55 days, about 60 days or longer. Insome embodiments, the fermentation conditions involve incubating theculture at a temperature of about 15° C. to about 40° C. In someembodiments, the fermentation conditions involve incubating the cultureat a temperature of about 15° C. to about 25° C., 20° C. to about 25°C., or about 18° C. to about 37° C. In some embodiments, thefermentation conditions involve incubating the culture at a temperatureof about 20° C.-25° C. In some embodiments, the fermentation process ofone or more fermentable sugars may be performed at a temperature ofabout 15° C., about 16° C., about 17° C., about 18° C., about 19° C.,about 20° C., about 21° C., about 22° C., about 23° C., about 24° C.,about 25° C., about 26° C., about 27° C., about 28° C., about 29° C.,about 30° C., about 31° C., about 32° C., about 33° C., about 34° C.,about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., orabout 40° C. In some embodiments, the fermentation conditions involveincubating the culture at a temperature of about 20° C.

Fermenting the cultures to produce a fermented beverage may be performedin any suitable vessel, such as a batch reactor, carboy, fermenter, orjar. Examples of vessels for fermentation include, without limitation,static batch reactors (e.g., plastic bucket, glass bucket, stainlesssteel batch reactor (e.g., 5 gallon, 50 gallon, 500 gallon, 1000gallon)), aerated batch reactors (e.g., vinegar fermenter, acetator),and more complex reactor, such as aerobic or anaerobic reactors, stirredor static reactors, batch or continuous reactors.

In some embodiments, one or more additional components, such asflavoring components, sweetening agents, or nutritive components, may beadded to the medium prior to or after the fermentation process. In someembodiments, one or more additional components, such as flavoringcomponents or nutritive components, may be added to the fermentedbeverage.

Various refinement, filtration, and processing steps may occursubsequent to fermenting the culture, after which the fermented beverageis bottled (e.g., captured and sealed in a container (e.g., glassbottle, aluminum can, etc) for distribution, storage, or consumption).Following bottling of the fermented beverages, minimal furtherfermentation may occur, however the levels of sugar, alcohol, andorganic acids are stable. Any of the methods described herein mayfurther involve pasteurizing and/or carbonating the fermented beverage.In some embodiments, the method further comprises carbonating thefermented beverage to produce a carbonated fermented beverage. Methodsof carbonating fermented beverages are known in the art and include, forexample, force carbonating with a gas (e.g., carbon dioxide, nitrogen),naturally carbonating by adding a further sugar source to the fermentedbeverage to promote further fermentation and production of carbondioxide (e.g., bottle conditioning). In some embodiments, the fermentedbeverage is not carbonated (e.g., still).

EXAMPLES Example 1: Use of Symbiotic Microbial Community to Produce anExemplary Fermented Beverage

To produce the fermented beverage, a base fermentation medium wasprepared containing 10 g/L cane sugar, 0.55% ginger w/w, and 0.2% v/vlemon juice (unconcentrated), with an initial pH of approximately 3.9.The media was sterilized by boiling for at least 30 minutes in a 5gallon stainless steel brewing container. The medium was allowed to coolto 30° C., and a starter culture of an exemplary symbiotic microbialcommunity containing microbial strains having 16S rDNA sequencesprovided by SEQ ID NO: 5, SEQ ID NO: 2, SEQ ID NO: 6, and SEQ NO: 7 wasthen added to the medium (e.g., 1:250 v/v for liquid starter culture oran equivalent in final mass equivalent freeze-dried starter culture).The inoculated medium was fermented at room temperature (approximately20° C.-22° C.) for 7-12 days, or until a final pH of approximately 2.8was achieved with approximately 3×10⁷ total CFU/mL. The final fermentedbeverage could then be flavored with spices or herbs, carbonated, andthen canned or bottled for distribution and consumption.

Final levels of sugar(s), alcohol, and organic acid(s) were analyzed inthe fermented beverage and compared to initial levels. The fermentedbeverages were also evaluated by blinded testers in a sensory panelbased on smell and taste discriminants, such as consistency, mouthfeel,texture, taste, sweetness, bitterness, yeast, mineral, acetone, acid,sugar, fruit and balance.

Symbiosis and Fermentation Rate

To evaluate whether the microbial communities provided an advantage inthe context of the rate of fermentation, the microbial community wasfermented as a community or as each of the individual strains (eachinoculated at 4-fold higher concentrations individually as compared toin combination in the community, such that the initial total microbialbiomass was equivalent across fermentations). The same fermentationprotocol was followed as described above with a 200 mL final volume andutilizing a 10 g/L (1%) cane sugar base with ginger. The initial sugarcontent was confirmed by enzymatic sugar testing for total sucrose,glucose, and fructose. Three distinct biological replicates wereperformed, and pH was determined daily by removing a 1 mL aliquot sampleof the fermentation and measuring pH with a calibrated pH meter.

The microbial community was found to have faster fermentation speed thanany of the individual strains (FIG. 1A). For example at day 3, the pH ofthe fermentation containing the microbial community was found to besignificantly lower than the pH of the fermentation with the strain thatbest dropped pH (Strain7, SEQ ID NO: 7) (using unpaired T-test,two-sided, p=0.0463). See, FIG. 1B. These data indicated that themicrobial community was symbiotic, and the microbial community had anincreased fermentation rate as compared to the individual microbialstrains.

Symbiosis and Biomass

To evaluate wherein the microbial communities provided an advantage inthe context of the end biomass using viable cell count as a proxy, themicrobial community was fermented as a community or with each of theindividual strains (each inoculated at 4-fold higher concentrationsindividually as compared to in combination in the community). The samefermentation protocol was followed as described above with a 200 mLfinal volume and utilizing a 10 g/L (1%) cane sugar base. The initialsugar content was confirmed by enzymatic sugar testing for totalsucrose, glucose, and fructose. Three distinct biological replicateswere performed, and viable cell counts were determined daily by removinga sample of the fermentation and using a standard plating assay byplating a fixed volume of liquid on an agar plate at different dilutionsand counting the number of colony forming units (CFUs).

The microbial community together showed higher end biomass at the end offermentation (FIG. 2A) and 7 days after the end of fermentation (FIG.2A). At 7 days after fermentation, Strain 6 (SEQ ID NO: 6) was notdetected above the assay limit of detection of 0.5×10⁴ CFUs/mL. The endbiomass of the microbial community was found to be significantly higher(effect size, ˜5.5 fold higher) than the biomass of the strain that hasthe highest end biomass (Strain 7, SEQ ID NO: 7) (using paired T test,across Day 0 and Day 7, two sided, p=0.0078). See, FIG. 2C. These dataindicated that the microbial community was symbiotic, and the microbialcommunity had an increased end biomass as compared to the individualmicrobial strains.

Microbial Communities are Compatible with Various Recipe Bases andConcentrations

The microbial communities were evaluated in fermentation processesinvolving different sugar bases. The same fermentation protocol wasfollowed as described above with a 200 mL final volume utilizingdifferent sugar bases (either “Ginger” containing cane sugar withginger, or “Apple” containing apple fruit juice) at different initialsugar concentrations including 5 g/L, 10 g/L, 15 g/L and 20 g/L sugarbase. The initial sugar content was confirmed by enzymatic sugar testingfor total sucrose, glucose, and fructose.

Three distinct biological replicates were performed, and variousmeasurements were performed including change in pH over time, communitybiomass, sugar profiling, and shelf stability assessments.

For each of the sugar bases and amounts tested, the microbial communitywas able to drop pH to below pH 3.0 within approximately 5-7 days (FIGS.3A and 3B). As shown in FIG. 4, the microbial communities were found toreach a high number of end CFUs/mL, in most conditions higher than 10⁷CFUs/mL. All fermentations reached more than 1 billion viable microbesper 8 fluid ounce serving on average.

In addition, the terminal sugar levels at the end of fermentation (e.g.,the time of bottling) and 14 days later (e.g., 14 days after bottling)was determined by enzymatic sugar assay for total sucrose, glucose, andfructose. As shown in FIG. 5A the residual sugar level increased basedon the initial sugar content in the different medium formulations. Sugarlevels slightly decreased 14 days after bottling. See, FIG. 5B. Theginger/sugar base at an initial level of 5 g/L resulted in a residuallevel of sugar below the threshold of 0.5 g of sugar per 8 oz servinglimit, allowing the product to be characterized as a 0-sugar beverage,according to the U.S. FDA Food Code. As shown in FIG. 5C the sugarlevels were significantly reduced from starting amounts in allconditions.

In addition, the shelf stability of the fermented beverages was assessedusing an accelerated 35° C. shelf stability assay. Briefly, fermentedbeverages were maintained at 35° C. elevated temperatures. Fermentedbeverages maintained at 35° C. for 5 days (across all sugarconcentrations and fruit/sugar bases) were indistinguishable fromsamples refrigerated immediately after fermentation in a blindedtaste/sensory panel. As shown in FIG. 6, the pH of the fermentedproducts remained stable when held at 35° C. at the time pointsassessed.

In addition, the longer term shelf stability of the fermented beverageswas assessed in an independent experiment following the protocoldescribed above. A base medium containing 10 g/L cane sugar with gingerwas utilized for the fermentation. The pH was analyzed over time forfermented beverages that were stored at room temperature (22° C.) andhigh temperature (35° C.). As shown in FIG. 7, the pH of the fermentedbeverages remained stable over the time assessed, more than 6 weeks.

In addition, the stability of microbial cultures in the fermentedbeverages was also assessed. A base media containing cane sugar withginger with sugar levels of 5 g/L, 10 g/L, 15 g/L and 20 g/L wereutilized for the fermentations, which were performed as described above.The biomass (CFUs/mL) of each fermented beverage was determined at theend of fermentation (day 0) and after 28 days (day 28) of storage atroom temperature. As shown in FIG. 8, while CFUs/mL decreased over time,nearly all fermented beverages retained on the order of billions of CFUsper 8 oz serving after the extended storage time.

Alcohol Content

The same fermentation protocol was followed as described above utilizingdifferent sugar bases (either “Ginger” containing cane sugar withginger, or “Apple” containing apple fruit juice) at different initialsugar concentrations including 5 g/L, 10 g/L, 15 g/L and 20 g/L sugarbase. The alcohol content (alcohol by volume (ABV)) was assessed for thefermented beverages by enzymatic enzyme assay following AOAC OfficialMethod 2019.08 (see, e.g., Ivory et al. J. AOAC Inter. (2020) 104(2):422-430), using a commercially available kit per the manufacturersrecommendations. As shown in FIG. 9, the ABV of each of the fermentedbeverages was low and near undetectable levels.

In sum, these data indicate that the microbial communities can be usedin fermentation processes using medium containing various formulations,such as juice and sugar bases, and result in successful fermentation,with high viable CFUs, while significantly reducing sugar, and beingshelf-stable.

Strain Stability in Fermented Beverages

The stability of the microbial strains in the fermented beverages wasalso evaluated over time. Fermentations were performed using a microbialcommunity or as individual strains. For the individual strainfermentations, each strain was inoculated at 4-fold higherconcentrations as compared to the combination in the community. Thefermentations were performed as described in Example 1 using a 200 mLfinal volume and a 10 g/L (1%) cane sugar base medium. The initial sugarcontent was confirmed by enzymatic sugar testing for total sucrose,glucose, and fructose. Three independent biological replicates wereperformed. Viable cell counts were determined over 80 days by removing asample of the fermentation and using a standard plating assay toenumerate colony forming units (CFUs) at each time point.

As shown in FIG. 10, the microbial strains when fermented as a communityresulted in significantly higher stability (i.e., higher CFUs/mL) overtime, as compared to the strains when fermented individually, indicatingsynergy in microbial stability over time in the fermented beverage.

Example 2: Use of Diverse Microbial Communities to Produce FermentedBeverages

It was observed that the combination of acetic acid bacterial strains(GROUP I) and lactic acid bacterial strains (GROUP II) led to desirableemergent characteristics with respect to sensory profile of thefermented beverage and ability of the microbial community to fermentsugar to acids better (faster, more efficiently) than individualmicrobial strains.

Additional example microbial communities were designed containing atleast one strain from GROUP I, at least one strain from GROUP II, andoptionally one or more accessory strains from GROUP III, see Table 1.Each complex community was assembled (in triplicate) on a microtiterplate in 80 μL following a similar protocol described in Example 1. Thechange in pH was assessed at various time points over the course of fourdays. Single-strain fermentations containing the individual microbialstrains (in triplicate) were performed.

The terminal pH was determined at 99.3 hours by taking the median acrossreplicates (with linear interpolation to account for minor offset inmeasurement times). The level of synergy in the microbial communities(i.e., symbiotically fermented sugar to acid more efficiently as amicrobial community as compared to the individual microbial strain) wasassessed by computing a “synergy score” at each timepoint. Specifically,the synergy score represents the difference in median pH of themicrobial community versus the minimal median pH of the individualmicrobial strains (with scores below zero corresponding to communitiesthat exhibit synergy, on a logarithmic scale). Table 1 shows grouping ofmicrobial strains used to generate the microbial communities describedherein. Table 2 shows the median terminal pH, as well as the synergyscore at the terminal timepoint, for all synergistic combinations.

TABLE 1 Microbial strains GROUP I GROUP II GROUP III Strain 1 (SEQ IDNO: 1) Strain 4 (SEQ ID NO: 4) Strain 8 (SEQ ID NO: 8) Strain 2 (SEQ IDNO: 2) Strain 5 (SEQ ID NO: 5) Strain 9 (SEQ ID NO: 9) Strain 3 (SEQ IDNO: 3) Strain 6 (SEQ ID NO: 6) Strain 10 (SEQ ID NO: 10) Strain 7 (SEQID NO: 7) Strain 11 (SEQ ID NO: 11) Strain 12 (SEQ ID NO: 12) Strain 13(SEQ ID NO: 13) Strain 14 (SEQ ID NO: 14) Strain 15 (SEQ ID NO: 15)Strain 16 (SEQ ID NO: 16) Strain 17 (SEQ ID NO: 17) Strain 18 (SEQ IDNO: 18) Strain 19 (SEQ ID NO: 19)

TABLE 2 Synergy scores and terminal pH of exemplary microbialcommunities Synergy Terminal pH (median (median n = 3 n = 3 Sequence IDsGroups replicates) replicates) SEQ 2, SEQ 4, SEQ GROUP I, GROUP II,−1.48 2.89 9 GROUP III SEQ 2, SEQ 4, SEQ GROUP I, GROUP II, −1.07 3.14 5GROUP II SEQ 1, SEQ 2, SEQ GROUP I, GROUP I, −0.51 2.86 2, SEQ 4, SEQ 5GROUP I, GROUP II, GROUP II SEQ 1, SEQ 2, SEQ GROUP I, GROUP I, −0.363.01 4, SEQ 5 GROUP II, GROUP II SEQ 1, SEQ 2, SEQ GROUP I, GROUP I,−0.33 3.04 6, SEQ 6 GROUP II, GROUP II SEQ 1, SEQ 4, SEQ GROUP I, GROUPII, −0.09 3.28 4, SEQ 4, SEQ 5 GROUP II, GROUP II, GROUP II SEQ 1, SEQ4, SEQ GROUP I, GROUP II, −0.09 3.28 4, SEQ 5 GROUP II, GROUP II SEQ 1,SEQ 4, SEQ GROUP I, GROUP II, −0.09 3.28 4, SEQ 5 GROUP II, GROUP II SEQ1, SEQ 4, SEQ GROUP I, GROUP II, −0.06 3.31 4, SEQ 5 GROUP II, GROUP IISEQ 1, SEQ 4, SEQ GROUP I, GROUP II, −0.06 3.31 4, SEQ 4, SEQ 5 GROUPII, GROUP II, GROUP II SEQ 1, SEQ 4, SEQ GROUP I, GROUP II, −0.06 3.314, SEQ 5 GROUP II, GROUP II SEQ 1, SEQ 4, SEQ GROUP I, GROUP II, −0.053.32 4, SEQ 5, SEQ 6 GROUP II, GROUP II, GROUP II SEQ 1, SEQ 4, SEQGROUP I, GROUP II, −0.05 3.32 4, SEQ 4, SEQ 5 GROUP II, GROUP II, GROUPII SEQ 1, SEQ 4, SEQ GROUP I, GROUP II, −0.05 3.32 4, SEQ 5, SEQ 6 GROUPII, GROUP II, GROUP II SEQ 1, SEQ 2, SEQ GROUP I, GROUP I, −0.04 2.81 3,SEQ 3, SEQ 6 GROUP I, GROUP I, GROUP II SEQ 3, SEQ 4, SEQ GROUP I, GROUPII, −0.03 2.82 4, SEQ 4, SEQ 5 GROUP II, GROUP II, GROUP II SEQ 1, SEQ4, SEQ GROUP I, GROUP II, −0.03 3.34 5, SEQ 5 GROUP II, GROUP II SEQ 1,SEQ 4, SEQ GROUP I, GROUP II, −0.02 2.88 4, SEQ 13 GROUP II, GROUP IIISEQ 1, SEQ 5, SEQ GROUP I, GROUP II, −0.02 2.88 13 GROUP III SEQ 1, SEQ4, SEQ GROUP I, GROUP II, −0.02 3.35 4, SEQ 5, SEQ 6 GROUP II, GROUP II,GROUP II SEQ 1, SEQ 5, SEQ GROUP I, GROUP II, −0.02 3.35 6, SEQ 6, SEQ11 GROUP II, GROUP II, GROUP III SEQ 2, SEQ 3, SEQ GROUP I, GROUP I,−0.01 2.84 4, SEQ 6, SEQ 7 GROUP II, GROUP II, GROUP II SEQ 1, SEQ 4,SEQ GROUP I, GROUP II, −0.01 3.36 5, SEQ 5 GROUP II, GROUP II SEQ 1, SEQ4, SEQ GROUP I, GROUP II, −0.01 3.36 4, SEQ 5, SEQ 5 GROUP II, GROUP II,GROUP II

Furthermore, fermentations using the microbial communities described inTable 1 were scaled up to produce a fermented beverage. Using thefermentation protocol described in Example 1, the microbial communitieswere fermented at 200 mL scales. At terminal fermentation, variousproperties of the fermented beverages were measured including totalsugar content, acetic acid level, gluconic acid level, and D-/L-lacticacid level, each determined by enzymatic sugar assays.

Table 3 shows data from the fermented beverages corresponding to themean of technical triplicate measurements. Each of the microbialcommunities significantly reduced the initial sugar content below theinitial 10 g/L level, and the fermented beverages contained high levelsof organic acids (e.g., gluconic, lactic acid). In addition, the aceticacid levels in the fermented beverages produced using the microbialcommunities were significantly lower than fermented beverages such askombucha produced using conventional methods, which are typicallybetween 4-8 g/L.

TABLE 3 Characteristics of fermented beverages produced using theexemplary microbial communities D-Lactic/ D- D- Total Acetic GluconicL-Lactic Sequence Sucrose Glucose Fructose Sugars Acid Acid AcidTerminal IDs Groups (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) pH SEQ 5,GROUP II, 0.97 0.27 2.69 4.55 0.62 0.82 1.41 2.82 SEQ 2, GROUP I, SEQ 6,GROUP II, SEQ 7 GROUP II SEQ 5, GROUP II, 1.39 0.35 3.18 5.79 0.12 0.860.03 2.69 SEQ 2, GROUP I, SEQ 4 GROUP II SEQ 2, GROUP I, 1.33 0.25 2.835.20 0.49 0.67 1.17 2.70 SEQ 1, GROUP I, SEQ 4, GROUP II, SEQ 5, GROUPII, SEQ 6, GROUP II, SEQ 7 GROUP II SEQ 5, GROUP II, 1.39 0.88 3.24 6.650.05 0.95 0.02 2.76 SEQ 2, GROUP I, SEQ 1 GROUP I SEQ 5, GROUP II, 1.340.31 3.43 5.92 0.53 0.86 1.08 2.72 SEQ 2, GROUP I, SEQ 1, GROUP I, SEQ4, GROUP II, SEQ 7 GROUP II SEQ 2, GROUP I, 1.48 0.21 2.88 5.41 0.400.67 1.10 2.77 SEQ 1, GROUP I, SEQ 4, GROUP II, SEQ 6, GROUP II, SEQ 7GROUP II SEQ 5, GROUP II, 1.56 0.19 2.65 5.27 0.53 0.56 1.09 2.73 SEQ 2,GROUP I, SEQ 4, GROUP II, SEQ 7 GROUP II SEQ 5, GROUP II, 0.23 0.94 3.715.46 0.14 1.84 1.00 2.82 SEQ 7, GROUP II, SEQ 9, GROUP III, SEQ 4, GROUPII, SEQ 3 GROUP I SEQ 7, GROUP II, 0.44 1.19 3.57 6.01 0.14 1.89 0.922.86 SEQ 9, GROUP III, SEQ 4, GROUP II, SEQ 3 GROUP I

Example 3: Resistance to Contaminating Microorganisms

To assess resilience of fermented beverages containing the microbialcommunities described herein to contaminating microorganisms, achallenge study was performed. In general, for a challenge study,fermentations are prepared as described in Example 1 containing amicrobial community or individual microbial strains. Undesiredmicroorganisms, such as those that may be found commonly in breweryenvironments (e.g., contaminants, pathogens such as Brettanomyces sp. orSaccharomyces sp.), are added to the fermentation at initialfermentation or at various time points during fermentation (for example,in the middle of fermentation, e.g., at day 3).

Here, microbial communities were fermented as a community (strains 2, 5,6, and 7) or an individual strains. For the individual strainfermentations, each strain was inoculated at 4-fold higherconcentrations as compared to the combination in the community. Thefermentations were performed as described in Example 1 using a 200 mLfinal volume and a 10 g/L (1%) cane sugar base medium. The initial sugarcontent was confirmed by enzymatic sugar testing for total sucrose,glucose, and fructose. Four to five independent biological replicateswere performed.

The undesired microorganism Brettanomyces sp. was introduced at a 1:100dilution at the initial fermentations. CFUs/mL of the Brettanomyces weredetermined by plating on selective enumeration media. As shown in FIG.11, the fermentations containing the microbial community displayedsignificant resilience to invasion as compared to the control and equalto or better than the several of the fermentations containing theindividual strains, demonstrating collective resilience conferred by themicrobial community.

Example 4: Analysis of Exemplary Fermented Product

A fermented beverage was produced using a community containing Strains2, 5, 6, and 7, as described in Example 1 using a medium containing 1%sugar and ginger. End organic acid profiles depend on factors such asthe starting medium formulation. All assays were enzymatic assays, andthe values shown below represent the average of three technicalreplicates:

1% sugar base with ginger

-   -   Acetic acid: 0.62 g/L    -   Gluconic acid: 0.82 g/L    -   D- and L-Lactic acid (sum): 1.41 g/L

Further fermented beverages were produced using a community containingStrains 2, 4, 5, and 7, as described in Example 1 using a mediumcontaining 1% sugar or 1% fruit juice (corresponding to a 10 g/L initialsugar level). Samples of the fermented beverage were further evaluatedusing full kombucha profile testing, including sugar profile analysis.See, Tables 4, 5, and 6.

TABLE 4 Results of kombucha profile analysis Test Method Result UnitsAlcohol by volume by AOAC 2016.12 0.02 Percent gas chromatograph TotalYeast Count AOAC 2014.05 None Detected CFUs/mL Total Mold Count AOAC2014.05 None Detected CFUs/mL Total Aerobic Bacteria AOAC 2015.13186,000,000 CFUs/mL Count Staphylococcus aureus AOAC 2003.07 NoneDetected CFUs/mL E. coli 3M Petrifilm None Detected CFUs/mL E.Coli/Coliform Count Plates pH ASBC Beer-9 2.86 Titratable Acidity AOAC942.15 1.14 g/L (Measured as Acetic Acid) Acetic Acid Enzymatic 0.74 g/LGluconic Acid Enzymatic 0.02 g/L Lactic Acid Enzymatic 0.05 g/L MalicAcid Enzymatic 1 mg/L

TABLE 5 Sugar profile of a fermented beverage produced using a mediumcontaining 1% sugar Analysis Sugar Profile Result Fructose 0.25% Glucose<0.10% Sucrose <0.10% Lactose <0.10% Maltose <0.10% Galactose <0.10%Total Sugar 0.25%

TABLE 6 Sugar profile of a fermented beverage produced using a mediumcontaining 1% fruit juice Analysis Sugar Profile Result Fructose 0.18%Glucose 0.13% Sucrose 0.14% Lactose <0.10% Maltose <0.10% Galactose<0.10% Total Sugar 0.45%

ENUMERATED EMBODIMENTS

1. A fermented beverage comprising

a symbiotic microbial community comprising at least one bacterial strainhaving a 16S rDNA sequence comprising at least 95% sequence identity toa nucleic acid sequence provided by SEQ ID NOs: 1-3, and at least onebacterial strain having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs:4-7, and at least one additional microbial strain,

a sugar content that is less than 20 grams per liter (g/L), and

an ethanol level that is less than 0.5% alcohol by volume (abv, v/v).

2. The fermented beverage of embodiment 1, wherein the symbioticmicrobial community comprises at least two, at least three, or at leastfour additional microbial strains.

3. The fermented beverage of embodiment 1 or 2, wherein at least one ofthe additional microbial strains is a bacterial strain.

4. The fermented beverage of any one of embodiments 1-3, wherein each ofthe additional microbial strains is a bacterial strain.

5. The fermented beverage of any one of embodiments 2-4, wherein theadditional bacterial strain belongs to the genus Lactobacillus,Gluconobacter, Leuconostoc, Acetobacter, Hafnia/Obesumbacterium,Lactococcus, Pediococcus, or Bacillus.

6. The fermented beverage of embodiment 5, wherein the additionalbacterial strain belongs to the species Acetobacter pasteurianus, A.ghanesis, A. orientalis, A. tropicalis, Gluconobacter oxydans, G.roseus, G. japonicus, G. frateurii, Leuconostoc mesenteroides,Lactobacillus senmaizukei, L. brevis, L. parakefiri, L. hilgardii, L.diolivorans, L. rapi, L. kisonesis, L. buchneri, L. fuchuensis, L.plantarum, L. paraplantarum, L. fabifermentans, L. pentosus, L.graminis, L. composti, Bacillus zanthoxyli, B. qingshengii, B.aryabhattai, B. flexus, B. megaterium, Hafnia alvei, Obesumbacteriumproteus, Lactococcus taiwanensis, L. lactis, Lactobacillus casei, L.paracasei, Pediococcus claussenii, P. stilesii, P. pentosaceus, P.acidilactici, Gluconacetobacter liquefaciens, Lactobacillus cerevisiae,L. kefiri, L. sunkii, L. otakiensis, L. parabuchneri, Leuconostoclactis, L. palmae, L. holzapfelii, L. citreum, Lactobacillus nagelii, L.satsumensis, Acetobacter papayae, A. suratthaniensis, A. peroxydans,Gluconacetobacter takamatsuzukensis, or G. asukensis.7. The fermented beverage of any one of embodiments 1-6, wherein theadditional bacterial strain has a 16S rDNA sequence comprising at least95% sequence identity to a nucleic acid sequence provided by SEQ ID NOs:8-19.8. The fermented beverage of any one of embodiments 1-7, wherein thesymbiotic microbial community comprises bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 4, 5, 6, 7, and 20-24.9. The fermented beverage of any one of embodiments 1-8, wherein thesymbiotic microbial community comprises bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 5, 6, and 7.10. The fermented beverage of any one of embodiments 1-7, wherein thesymbiotic microbial community comprises bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 4, and 5.11. The fermented beverage of any one of embodiments 1-9, wherein thesymbiotic microbial community consists of bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 5, 6, and 7.12. The fermented beverage of any one of embodiments 1-8 and 10, whereinthe symbiotic microbial community consists of bacterial strains having a16S rDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 4, and 5.13. The fermented beverage of any one of embodiments 1-9, wherein thesymbiotic microbial community comprises bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 20, 22, and 24.14. The fermented beverage of any one of embodiments 1-8 and 10, whereinthe symbiotic microbial community comprises bacterial strains having a16S rDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 23, and 20.15. The fermented beverage of any one of embodiments 1-9 and 13, whereinthe symbiotic microbial community consists of bacterial strains having a16S rDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 20, 22, and 24.16. The fermented beverage of any one of embodiments 1-8, 10, and 14,wherein the symbiotic microbial community consists of bacterial strainshaving a 16S rDNA sequence comprising at least 95% sequence identity toa nucleic acid sequence provided by SEQ ID NOs: 21, 23, and 20.17. The fermented beverage of any one of embodiments 1-8, wherein thesymbiotic microbial community comprises bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 4, 5, and 7.18. The fermented beverage of any one of embodiments 1-8 and 17, whereinthe symbiotic microbial community consists of bacterial strains having a16S rDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 4, 5, and 7.19. The fermented beverage of any one of embodiments 1-8 or 17, whereinthe symbiotic microbial community comprises bacterial strains having a16S rDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 23, 20, and 24.20. The fermented beverage of any one of embodiments 1-8, 17, and 19,wherein the symbiotic microbial community consists of bacterial strainshaving a 16S rDNA sequence comprising at least 95% sequence identity toa nucleic acid sequence provided by SEQ ID NOs: 21, 23, 20, and 24.21. The fermented beverage of any one of embodiments 1-20, wherein thesymbiotic microbial community does not comprise a yeast strain.22. The fermented beverage of any one of embodiments 1-21, wherein thesymbiotic microbial community comprises at least 2×10⁷ colony formingunits of each bacterial strain and each additional microbial strain permilliliter of the fermented beverage.23. The fermented beverage of any one of embodiments 1-22, comprising alevel of acetic acid less than 1 gram per liter (g/L).24. The fermented beverage of any one of embodiments 1-23, comprising alevel of organic acid that is greater than 1 gram per liter (g/L),wherein the organic acid is not acetic acid.25. The fermented beverage of embodiment 24, wherein the organic acid islactic acid, gluconic acid, ketogluconic acid, or a combination thereof.26. The fermented beverage of any one of embodiments 1-25, wherein thefermented beverage is shelf stable for at least 2 weeks at a temperatureof about 20° C.27. The fermented beverage of any one of embodiments 1-26, wherein thefermented beverage is shelf stable for at least 1 week at a temperatureof about 40° C.28. The fermented beverage of any one of embodiments 1-27, wherein thepH of the fermented beverage is less than about 3.5.29. The fermented beverage of any one of embodiments 1-28, wherein thefermented beverage is kombucha, seltzer, soda, gut shot, water kefir,jun, fruit juice, vegetable juice, ginger beer, a flavored waterproduct, or a probiotic beverage.30. The fermented beverage of any one of embodiments 1-29, wherein atleast one of the microbial strains is derived from a fermented foodproduct.31. The fermented beverage of any one of embodiments 1-30, wherein eachof the microbial strains is derived from a fermented food product.32. The fermented beverage of any one of embodiments 1-31, wherein thebacterial strains and the additional microbial strains are live in thefermented beverage.33. The fermented beverage of any one of embodiments 1-32, furthercomprising one or more additional components.34. The fermented beverage of embodiment 33, wherein the additionalcomponent is a vitamin, mineral, or flavoring additive.35. The fermented beverage of embodiment 33 or 34, wherein the one ormore additional component is selected from the group consisting of blacktea, green tea, fruit juice, and vegetable juice.36. A method of producing a fermented food beverage, comprising

(i) providing a medium comprising a fermentable sugar at an initialsugar level;

(ii) adding a symbiotic microbial community to the medium to produce aculture, wherein the symbiotic microbial community comprises at leastone bacterial strain having a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs:1-3, and at least one bacterial strain having a 16S rDNA sequencecomprising at least 95% sequence identity to a nucleic acid sequenceprovided by SEQ ID NOs: 4-7, and at least one additional microbialstrain; and

(iii) fermenting the culture under conditions to produce a fermentedbeverage.

37. The method of embodiment 36, wherein the symbiotic microbialcommunity comprises at least two, at least three, or at least fouradditional microbial strains.

38. The method of embodiment 36 or 37, wherein at least one of theadditional microbial strains is a bacterial strain.

39. The method of any one of embodiments 36-38, wherein each of theadditional microbial strains is a bacterial strain.

40. The method of any one of embodiments 36-40, wherein the additionalbacterial strain belongs to the genus Lactobacillus, Gluconobacter,Leuconostoc, Acetobacter, Hafnia/Obesumbacterium, Lactococcus,Pediococcus, or Bacillus.

41. The method of embodiment 40, wherein the additional bacterial strainbelongs to the species Acetobacter pasteurianus, A. ghanesis, A.orientalis, A. tropicalis, Gluconobacter oxydans, G. roseus, G.japonicus, G. frateurii, Lactobacillus senmaizukei, L. brevis,Leuconostoc mesenteroides, Lactobacillus parakefiri, L. hilgardii, L.diolivorans, L. rapi, L. kisonesis, L. buchneri, L. fuchuensis, L.plantarum, L. paraplantarum, L. fabifermentans, L. pentosus, L.graminis, L. composti, Bacillus zanthoxyli, B. qingshengii, B.aryabhattai, B. flexus, B. megaterium, Hafnia alvei, Obesumbacteriumproteus, Lactococcus taiwanensis, L. lactis, Lactobacillus casei, L.paracasei, Pediococcus claussenii, P. stilesii, P. pentosaceus, P.acidilactici, Gluconacetobacter liquefaciens, L. cerevisiae, L. kefiri,L. sunkii, L. otakiensis, L. parabuchneri, Leuconostoc lactis, L.palmae, L. holzapfelii, L. citreum, Lactobacillus nagelii, L.satsumensis, Acetobacter papayae, A. suratthaniensis, A. peroxydans,Gluconacetobacter takamatsuzukensis, or G. asukensis.42. The method of any one of embodiments 36-41, wherein the additionalbacterial strain has a 16S rDNA sequence comprising at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NOs:8-19.43. The method of any one of embodiments 36-42, wherein the symbioticmicrobial community comprises bacterial strains having a 16S rDNAsequence comprising at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 2, 4, 5, 6, 7, and 20-24.44. The method of any one of embodiments 36-43, wherein the symbioticmicrobial community comprises bacterial strains having a 16S rDNAsequence comprising at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 2, 5, 6, and 7.45. The method of any one of embodiments 36-43, wherein the symbioticmicrobial community comprises bacterial strains having a 16S rDNAsequence comprising at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 2, 4, and 5.46. The method of any one of embodiments 36-44, wherein the symbioticmicrobial community consists of bacterial strains having a 16S rDNAsequence comprising at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 2, 5, 6, and 7.47. The method of any one of embodiments 36-43 and 45, wherein thesymbiotic microbial community consists of bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 4, and 5.48. The method of any one of embodiments 36-44, wherein the symbioticmicrobial community comprises bacterial strains having a 16S rDNAsequence comprising at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 21, 20, 22, and 24.49. The method of any one of embodiments 36-43 and 45, wherein thesymbiotic microbial community comprises bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 23, and 20.50. The method of any one of embodiments 36-44 and 48, wherein thesymbiotic microbial community consists of bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 20, 22, and 24.51. The method of any one of embodiments 36-43, 45, and 49, wherein thesymbiotic microbial community consists of bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 23, and 20.52. The method of any one of embodiments 36-43, wherein the symbioticmicrobial community comprises bacterial strains having a 16S rDNAsequence comprising at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 2, 4, 5, and 7.53. The method of any one of embodiments 36-43 and 52, wherein thesymbiotic microbial community consists of bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 2, 4, 5, and 7.54. The method of any one of embodiments 36-43 or 52, wherein thesymbiotic microbial community comprises bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 23, 20, and 24.55. The method of any one of embodiments 36-43, 52, and 54, wherein thesymbiotic microbial community consists of bacterial strains having a 16SrDNA sequence comprising at least 95% sequence identity to a nucleicacid sequence provided by SEQ ID NOs: 21, 23, 20, and 24.56. The method of any one of embodiments 36-55, wherein the symbioticmicrobial community does not comprise a yeast strain.57. The method of any one of embodiments 36-56, wherein the symbioticmicrobial community comprises at least 2×10⁷ colony forming units ofeach bacterial strain and each additional microbial strain permilliliter of the fermented beverage.58. The method of any one of embodiments 36-57, comprising a level ofacetic acid less than 1 gram per liter (g/L).59. The method of any one of embodiments 36-58, comprising a level oforganic acid that is greater than 1 gram per liter (g/L), wherein theorganic acid is not acetic acid.60. The method of embodiment 59, wherein the organic acid is lacticacid, gluconic acid, ketogluconic acid, or a combination thereof.61. The method of any one of embodiments 36-60, wherein the fermentedbeverage is shelf stable for at least 2 weeks at a temperature of about20° C.62. The method of any one of embodiments 36-61, wherein the fermentedbeverage is shelf stable for at least 1 week at a temperature of about40° C.63. The method of any one of embodiments 36-62, wherein the pH of thefermented beverage is less than about 3.5.64. The method of any one of embodiments 36-63, wherein the fermentedbeverage is kombucha, seltzer, soda, gut shot, water kefir, jun, fruitjuice, vegetable juice, ginger beer, a flavored water product, or aprobiotic beverage.65. The method of any one of embodiments 36-64, wherein at least one ofthe microbial strains is derived from a fermented food product.66. The method of any one of embodiments 36-65, wherein each of themicrobial strains is derived from a fermented food product.67. The method of any one of embodiments 36-66, wherein the bacterialstrains and the additional microbial strains are live in the fermentedbeverage.68. The method of any one of embodiments 36-67, further comprising oneor more additional components.69. The method of embodiment 68, wherein the additional component is avitamin, mineral, or flavoring additives.70. The method of embodiment 68 or 69, wherein the one or moreadditional component is selected from the group consisting of black tea,green tea, fruit juice, and vegetable juice.71. The method of any one of embodiments 36-70, wherein the fermentedbeverage has a pH less than about 3.5.72. The method of any one of embodiments 36-71, wherein the fermentingis performed in a batch reactor.73. The method of any one of embodiments 36-72, wherein the fermentingis performed at about 18-37° C.74. The method of any one of embodiments 36-73, wherein the fermentingis performed for at least 6 days at about 20° C.75. The method of any one of embodiments 36-74, wherein the initialsugar level is 2.5-20 grams per liter (g/L) of a sugar source.76. The method of any one of embodiments 36-75, wherein the initialsugar level is about 10 grams per liter (g/L) of a sugar source.76. The method of embodiment 76, wherein the sugar source is a canesugar, palm sugar, maple syrup, fruit juice, vegetable juice, brownsugar, molasses, agave nectar, honey, date syrup, date paste, datesugar, coconut sugar, or coconut water.77. The method of any one of claims 36-76, wherein the bacterial strainsand the additional microbial strain(s) replicate faster when in thesymbiotic microbial community compared to when not in the symbioticmicrobial community.78. The method of any one of embodiments 36-77, wherein the bacterialstrains and the additional microbial strain(s) grow to a higher biomasswhen in the symbiotic microbial community compared to when not in thesymbiotic microbial community.79. The method of any one of embodiments 36-78, wherein the symbioticmicrobial community comprises at least 2×10⁵ colony forming units.80. The method of any one of embodiments 36-79, wherein the biomass ofthe symbiotic microbial community is stable over at least 60 days.81. The method of any one of embodiments 36-80, wherein the symbioticmicrobial community reduces or prevents growth of undesired microbialstrains.82. The method of any one of embodiments 36-81, further comprisingcarbonating the fermented beverage to produce a carbonated fermentedbeverage.83. A fermented beverage obtained or obtainable by the method of any oneof embodiments 36-82.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of” “only one of” or“exactly one of” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A fermented beverage comprising a symbioticmicrobial community comprising a bacterial strain having a 16S rDNAsequence having at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NO: 2, a bacterial strain having a 16S rDNAsequence having at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NO: 4 or SEQ ID NO: 5, and an additionalmicrobial strain, a sugar content that is less than 20 grams per liter(g/L), an ethanol level that is less than 0.5% alcohol by volume (abv,v/v), and an acetic acid level that is less than 1 g/L.
 2. The fermentedbeverage of claim 1, wherein the symbiotic microbial community comprisesa bacterial strain having a 16S rDNA sequence having at least 95%sequence identity to a nucleic acid sequence provided by SEQ ID NO: 2, abacterial strain having a 16S rDNA sequence having at least 95% sequenceidentity to a nucleic acid sequence provided by SEQ ID NO: 4, and anadditional microbial strain.
 3. The fermented beverage of claim 1,wherein the symbiotic microbial community comprises a bacterial strainhaving a 16S rDNA sequence having at least 95% sequence identity to anucleic acid sequence provided by SEQ ID NO: 2, a bacterial strainhaving a 16S rDNA sequence having at least 95% sequence identity to anucleic acid sequence provided by SEQ ID NO: 5, and an additionalmicrobial strain.
 4. The fermented beverage of claim 1, wherein thesymbiotic microbial community comprises three bacterial strains having16S rDNA sequences of at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 2, 4 and
 5. 5. The fermented beverageof claim 1, wherein the symbiotic microbial community consists of abacterial strain having a 16S rDNA sequence having at least 95% sequenceidentity to a nucleic acid sequence provided by SEQ ID NO: 2, abacterial strain having a 16S rDNA sequence having at least 95% sequenceidentity to a nucleic acid sequence provided by SEQ ID NO: 4 or SEQ IDNO: 5, and an additional microbial strain.
 6. The fermented beverage ofclaim 1, wherein the symbiotic microbial community consists of abacterial strain having a 16S rDNA sequence having at least 95% sequenceidentity to a nucleic acid sequence provided by SEQ ID NO: 2, abacterial strain having a 16S rDNA sequence having at least 95% sequenceidentity to a nucleic acid sequence provided by SEQ ID NO: 4, and anadditional microbial strain.
 7. The fermented beverage of claim 1,wherein the symbiotic microbial community consists of a bacterial strainhaving a 16S rDNA sequence having at least 95% sequence identity to anucleic acid sequence provided by SEQ ID NO: 2, a bacterial strainhaving a 16S rDNA sequence having at least 95% sequence identity to anucleic acid sequence provided by SEQ ID NO: 5, and an additionalmicrobial strain.
 8. The fermented beverage of claim 1, wherein thesymbiotic microbial community comprises three bacterial strains having16S rDNA sequences of at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NOs: 2, 4 and
 5. 9. The fermented beverageof claim 1, wherein the symbiotic microbial community comprises abacterial strain having a 16S rDNA sequence having at least 97% sequenceidentity to a nucleic acid sequence provided by SEQ ID NO: 2, abacterial strain having a 16S rDNA sequence having at least 97% sequenceidentity to a nucleic acid sequence provided by SEQ ID NO: 4 or SEQ IDNO: 5, and an additional microbial strain.
 10. The fermented beverage ofclaim 1, wherein the symbiotic microbial community comprises a bacterialstrain having a 16S rDNA sequence having at least 97% sequence identityto a nucleic acid sequence provided by SEQ ID NO: 2, a bacterial strainhaving a 16S rDNA sequence having at least 97% sequence identity to anucleic acid sequence provided by SEQ ID NO: 4, and an additionalmicrobial strain.
 11. The fermented beverage of claim 1, wherein thesymbiotic microbial community comprises a bacterial strain having a 16SrDNA sequence having at least 97% sequence identity to a nucleic acidsequence provided by SEQ ID NO: 2, a bacterial strain having a 16S rDNAsequence having at least 97% sequence identity to a nucleic acidsequence provided by SEQ ID NO: 5, and an additional microbial strain.12. The fermented beverage of claim 1, wherein the symbiotic microbialcommunity comprises a bacterial strain having a 16S rDNA sequence havingat least 99% sequence identity to a nucleic acid sequence provided bySEQ ID NO: 2, a bacterial strain having a 16S rDNA sequence having atleast 99% sequence identity to a nucleic acid sequence provided by SEQID NO: 4 or SEQ ID NO: 5, and an additional microbial strain.
 13. Thefermented beverage of claim 1, wherein the symbiotic microbial communitycomprises a bacterial strain having a 16S rDNA sequence having at least99% sequence identity to a nucleic acid sequence provided by SEQ ID NO:2, a bacterial strain having a 16S rDNA sequence having at least 99%sequence identity to a nucleic acid sequence provided by SEQ ID NO: 4,and an additional microbial strain.
 14. The fermented beverage of claim1, wherein the symbiotic microbial community comprises a bacterialstrain having a 16S rDNA sequence having at least 99% sequence identityto a nucleic acid sequence provided by SEQ ID NO: 2, a bacterial strainhaving a 16S rDNA sequence having at least 99% sequence identity to anucleic acid sequence provided by SEQ ID NO: 5, and an additionalmicrobial strain.
 15. The fermented beverage of claim 1, wherein thefermented beverage does not comprise a yeast strain.
 16. The fermentedbeverage of claim 1, wherein in the pH of the fermented beverage is lessthan about 4.5.
 17. The fermented beverage of claim 1, wherein thefermented beverage is a seltzer, soda, flavored water product, or aprobiotic beverage.
 18. The fermented beverage of claim 1, wherein atleast one of the bacterial strains is derived from a fermented foodproduct.
 19. The fermented beverage of claim 1, wherein each of thebacterial strains is derived from a fermented food product.
 20. Thefermented beverage of claim 1, wherein each of the bacterial strains islive in the fermented beverage.
 21. The fermented beverage of claim 1,comprising between 3×10⁶ and 3×10⁸ colony forming units (CFUs) perbacterial strain per milliliter of fermented beverage.
 22. The fermentedbeverage of claim 1, comprising between 1.5×10⁷ and 1.5×10⁹ total CFUper milliliter of fermented beverage.
 23. The fermented beverage ofclaim 1, further comprising one or more additional components selectedfrom the group consisting of a nutritive component, a flavoringcomponent, a sweetening component and combinations thereof.
 24. Thefermented beverage of claim 23, wherein the nutritive component is avitamin or a mineral; and/or wherein the flavoring component is anatural fruit flavoring, a naturally derived fruit flavoring, or asynthetic fruit flavoring.
 25. The fermented beverage of claim 1,wherein the fermented beverage is shelf-stable, for at least two weeks,at a temperature of about 20° C. to about 22° C., whereinshelf-stability is assessed by viability of bacterial strains and/orflavor profile.
 26. The fermented beverage of claim 1, wherein thefermented beverage comprises a biomass that decreases by less than 10%over a period of 60 days at a temperature of about 20° C.-22° C.
 27. Thefermented beverage of claim 1, wherein the fermented beverage furthercomprises lactic acid, gluconic acid, ketogluconic acid, or acombination thereof.
 28. The fermented beverage of claim 1, wherein thesymbiotic microbial community reduces or prevents growth of one or moreundesired microbial strains.
 29. The fermented beverage of claim 1,wherein the fermented beverage is produced by a method comprising (i)providing a medium comprising a fermentable sugar at an initial sugarlevel; (ii) adding the symbiotic microbial community to the medium toproduce a culture; and (iii) fermenting the culture under conditions toproduce a fermented beverage.
 30. A method of producing the fermentedbeverage of claim 1, comprising (i) providing a medium comprising afermentable sugar at an initial sugar level; (ii) adding a symbioticmicrobial community to the medium to produce a culture, wherein thesymbiotic microbial community comprises a bacterial strain having a 16SrDNA sequence having at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NO: 2, a bacterial strain having a 16S rDNAsequence having at least 95% sequence identity to a nucleic acidsequence provided by SEQ ID NO: 4 or SEQ ID NO: 5, and an additionalmicrobial strain; and (iii) fermenting the culture under conditions toproduce a fermented beverage.